Methods and Compositions for the Selection of A Transgenic Plant

ABSTRACT

Compositions and methods are provided to screen, identify, select, isolate, and/or regenerate targeted integration events using seed priming. Seed priming provides the identification of a seed having stably incorporated into its genome a site-specific recombinase mediated integration of a selectable marker at a target locus operably linked to a promoter active in the seed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/559,022 filed Nov. 11, 2006, and claims the benefit of U.S.Application Ser. No. 60/784,098 filed Mar. 20, 2006, which are eachherein incorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to the genetic modification of plants. Inparticular, methods and compositions are provided for the selection oftransgenic plants.

BACKGROUND

The frequency of integration of a polynucleotide into a desiredchromosomal position is low, requiring the generation and screening oflarge numbers of progeny. Screening is typically time and laborintensive. Methods to improve the selection and screening process areneeded. Methods to improve the efficiency of screening for targetedintegration events in plants are needed. The present invention providesmethods and compositions using seed priming with targeted integrationsystems, providing increased efficiency for screening and/or selectionof targeted events.

SUMMARY

Compositions and methods are provided to screen, identify, select,isolate, and/or regenerate targeted integration events using seedpriming. Seed priming provides the identification of a seed havingstably incorporated into its genome a site-specific recombinase-mediatedintegration of a selectable marker at a target locus operably linked toa promoter active in the seed.

DETAILED DESCRIPTION

Compositions and methods to prime seeds include, but are not limited tothe following:

-   1. A method to identify a plant having a DNA construct stably    incorporated in its genome comprising:    -   (a) providing a population of seeds, wherein at least one seed        in the population has stably incorporated into its genome the        DNA construct comprising the following operably linked        components in the following order: a promoter active in the        seed, a first recombination site, a polynucleotide encoding a        selection marker that confers resistance to a selective agent,        and a second recombination site;    -   (b) contacting the population of seeds with a priming matrix        comprising an effective concentration of the selection agent for        a time sufficient to produce a population of primed seeds; and,    -   (c) incubating the population of primed seed under germination        conditions, thereby identifying plants having the DNA construct        stably incorporated into their genome.-   2. The method of 1, wherein the first and the second recombination    sites are dissimilar and non-recombinogenic with each other.-   3. The method of any one of 1 or 2, wherein the selective agent    comprises an herbicide, a growth regulator, or an antibiotic.-   4. The method of any one of 1-3, wherein the selective agent is    selected from the group consisting of glufosinate, phosphinothricin,    glyphosate, bromoxynil, methotrexate, imidazolinones, sulfonylureas,    cyanamide, kanamycin, G418, neomycin, and hygromycin B.-   5. The method of any one of 1-3, wherein the selection marker is    selected from the group consisting of Bar, phosphinothricin    acetyltransferase (PAT), glyphosate oxidoreductase (GOX),    5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), CP4, glyphosate    N-acetyltransferase (GAT), bromoxynil nitrilase (BXN), dihydrofolate    reductase, acetolactate synthase (ALS), cyanamide hydratase (CAH),    neomycin phosphotransferase (nptII), aminoglycoside    3′-phosphotransferase (APH3′ II), and hygromycin phosphotransferase    (hph).-   6. The method of any one of 1-5, wherein the selective agent is    cyanamide or an active derivative of cyanamide.-   7. The method of any one of 1-6, wherein the selection marker is    cyanamide hydratase.-   8. The method of any one of 1-7, wherein the polynucleotide encoding    the selection marker has been synthesized using plant preferred    codons.-   9. The method of 8, wherein the polynucleotide encoding the    selection marker has been synthesized using maize preferred codons.-   10. The method of any one of 1-9, wherein the DNA construct    comprises in the following order: a first expression unit comprising    the promoter active in the seed operably linked to the first    recombination site operably linked to the polynucleotide encoding    the selection marker; and a second expression unit comprising in the    following order, a second promoter active in the plant operably    linked to a polynucleotide of interest, and the second recombination    site.-   11. The method of any one of 1-9, wherein the DNA construct    comprises in the following order: a first expression unit comprising    the promoter active in the seed operably linked to the first    recombination site operably linked to the polynucleotide encoding    the selection marker; a second expression unit comprising in the    following order, a second promoter active in the plant operably    linked to a polynucleotide of interest; and a third expression unit    comprising in the following order a third promoter active in the    plant operably linked to the second recombination site.-   12. The method of any of 1-11, wherein the DNA construct comprises    the following operably linked components in the following order: the    promoter active in the seed; an ATG start codon; the first    recombination site; and the polynucleotide encoding the selection    marker, wherein the polynucleotide encoding the selection marker    lacks an operably linked promoter and an ATG start codon.-   13. The method of any of 1-9, wherein the DNA construct comprises in    the following order: a first expression unit comprising the    following operably linked components, the promoter active in the    seed, an ATG start codon, the first recombination site, and the    polynucleotide encoding the selection marker, wherein the    polynucleotide encoding the selection marker lacks an operably    linked promoter and an ATG start codon; a second expression unit    comprising in the following order, a second promoter active in the    plant operably linked to a polynucleotide of interest; and, a third    expression unit comprising in the following order, a third promoter    active in the plant operably linked to the second recombination    site.-   14. The method of any one of 1-9, wherein the DNA construct    comprises in the following order: a first expression unit comprising    the following operably linked components, the promoter active in the    seed, an ATG start codon, the first recombination site, and the    polynucleotide encoding the selection marker, wherein the    polynucleotide encoding the selection marker lacks an operably    linked promoter and an ATG start codon; a second expression unit    comprising in the following order, a second promoter active in the    plant operably linked to a polynucleotide of interest; and, a third    expression unit comprising, in the following order, a third promoter    operably linked to a second ATG start codon operably linked to the    second recombination site.-   15. The method of any one of 1-14, wherein the plant further has    stably incorporated into its genome a polynucleotide encoding a site    specific recombinase.-   16. The method of 15, wherein the site specific recombinase    comprises a FLP recombinase, a Cre recombinase, a lambda integrase,    a SSV1 integrase, or a phiC31 integrase.-   17. The method of any one of 1-16, wherein at least one of the first    or the second recombination sites are selected from the group    consisting of a lox site, a mutant lox site, a FRT site, a mutant    FRT site, an att site, and a mutant att site.-   18. The method of 2, wherein the first and the second recombination    site comprise a FRT site and a mutant FRT site.-   19. The method of 2, wherein the first and the second recombination    sites comprise mutant FRT sites.-   20. The method of any one of 1-19, wherein the plant is a monocot.-   21. The method of any one of 1-19, wherein the plant is a dicot.-   22. The method of any one of 20 or 21, wherein the plant is maize,    wheat, rice, sorghum, rye, oat, barley, millet, Brassica, alfalfa,    sunflower, safflower, soybean, tobacco, cotton, or Arabidopsis.-   23. A method of priming a seed comprising    -   (a) providing a seed having stably incorporated into its genome        a DNA construct comprising, in the following order, a promoter        active in the seed operably linked to a first recombination site        operably linked to a polynucleotide encoding a selection marker        that confers resistance to a selective agent; and    -   (b) contacting the seed with a priming matrix comprising an        effective concentration of the selective agent for a time        sufficient to produce a primed seed.-   24. The method of 23, wherein the DNA construct comprises in the    following order the promoter operably linked to the first    recombination site operably linked the polynucleotide encoding the    selection marker, and a second recombination site.-   25. The method of 24, wherein the first and the second recombination    sites are dissimilar and non-recombinogenic with each other.-   26. The method of any one of 23-25, further comprising drying the    primed seed.-   27. The method of any one of 23-26, further comprising incubating    the primed seed under germinating conditions.-   28. The method of any one of 23-27, wherein the selective agent    comprises an herbicide, a growth regulator, or an antibiotic.-   29. The method of any one of 23-28, wherein the selective agent is    selected from the group consisting of glufosinate, phosphinothricin,    glyphosate, bromoxynil, methotrexate, imidazolinones, sulfonylureas,    cyanamide, kanamycin, G418, neomycin, and hygromycin B.-   30. The method of 29, wherein the selection marker is selected from    the group consisting of Bar, phosphinothricin acetyltransferase    (PAT), glyphosate oxidoreductase (GOX),    5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), CP4, glyphosate    N-acetyltransferase (GAT), bromoxynil nitrilase (BXN), dihydrofolate    reductase, acetolactate synthase (ALS), cyanamide hydratase (CAH),    neomycin phosphotransferase (nptII), aminoglycoside    3′-phosphotransferase (APH3′ II), and hygromycin phosphotransferase    (hph).-   31. The method of any one of 23-30, wherein the selective agent is    cyanamide or an active derivative thereof.-   32. The method of any one of 23-31, wherein the selection marker is    cyanamide hydratase.-   33. The method of any one of 24-32, wherein the DNA construct    comprises in the following order: a first expression unit comprising    the following operably linked components, the promoter active in the    seed, the first recombination site, and the polynucleotide encoding    the selection marker that confers resistance to the selective agent;    and, a second expression unit comprising in the following order, a    second promoter active in a plant operably linked to a    polynucleotide of interest, and the second recombination site.-   34. The method of any one of 24-32, wherein the DNA construct    comprises in the following order: a first expression unit comprising    the following operably linked components, the promoter active in the    seed, the first recombination site, and the polynucleotide encoding    the selection marker; a second expression unit comprising in the    following order, a second promoter active in a plant operably linked    to a polynucleotide of interest; and, a third expression unit    comprising in the following order a third promoter active in the    plant operably linked to the second recombination site.-   35. The method of any one of 33-34, wherein the polynucleotide of    interest encodes a second selection marker.-   36. The method of 35, wherein the second selection marker confers    resistance to a second selective agent.-   37. The method of any one of 23-36, wherein the DNA construct    comprises in the following order: a first expression unit comprising    the following operably linked components, a promoter active in the    seed, an ATG start codon, the first recombination site, and the    polynucleotide encoding the selection marker, wherein the    polynucleotide encoding the selection marker lacks an operably    linked promoter and an ATG start codon.-   38. The method of any one of 24-36, wherein the DNA construct    comprises in the following order: a first expression unit comprising    the following operably linked components, a promoter active in the    seed, an ATG start codon, the first recombination site, and the    polynucleotide encoding the selection marker, wherein the    polynucleotide encoding the selection marker lacks an operably    linked promoter and an ATG start codon; a second expression unit    comprising, in the following order, a second promoter active in a    plant operably linked to a polynucleotide of interest; and, a third    expression unit comprising, in the following order, a third promoter    active in the plant operably linked to the second recombination    site.-   39. The method of any one of 24-36, wherein the DNA construct    comprises in the following order: a first expression unit comprising    the following operably linked components, a promoter active in the    seed, an ATG start codon, the first recombination site, and the    polynucleotide encoding the selection marker, wherein the    polynucleotide encoding the selection marker lacks an operably    linked promoter and an ATG start codon; a second expression unit    comprising in the following order, a second promoter active in a    plant operably linked to a polynucleotide of interest; and, a third    expression unit comprising in the following order, a third promoter    active in the plant operably linked to a second ATG start codon    operably linked to the second recombination site.-   40. The method of any one of 23-36, wherein the seed further has    stably incorporated into its genome a polynucleotide encoding a site    specific recombinase.-   41. The method of 40, wherein the site specific recombinase    comprises a FLP recombinase, a Cre recombinase, a lambda integrase,    a SSV1 integrase, or a phiC31 integrase.-   42. The method of any one of 24-25, wherein at least one of the    first or the second recombination sites are selected from the group    consisting of a lox site, a mutant lox site, a FRT site, a mutant    FRT site, an att site, and a mutant att site.-   43. The method of any one of 24-25, wherein the first and the second    recombination site comprise a FRT site and a mutant FRT site.-   44. The method of any one of 24-45, wherein the first and the second    recombination site comprise mutant FRT sites.-   45. The method of any one of 23-44, wherein the seed is from a    monocot.-   46. The method of 45, wherein the monocot is maize, sorghum, barley,    rice, oat, wheat, or millet.-   47. The method of any one of 23-44, wherein the seed is from a    dicot.-   48. A method to identify a plant having a targeted insertion of a    polynucleotide of interest in its genome comprising:    -   (a) providing a population of plants having stably incorporated        into their genome a DNA construct comprising a promoter active        in a seed operably linked to a target site, the target site        comprising a first recombination site;    -   (b) introducing into the population of plants a transfer        cassette comprising in the following order the first        recombination site operably linked to a polynucleotide encoding        a selection marker that confers resistance to a selective agent,        wherein the polynucleotide is not operably linked to a promoter,        and the polynucleotide of interest;    -   (c) providing a recombinase to the population of plants, wherein        the recombinase recognizes and implements recombination at the        first recombination site;    -   (d) contacting a population of seeds produced from the        population of plants from step (c) with a priming matrix        comprising an effective concentration of the selective agent for        a time sufficient to produce a population of primed seeds; and,    -   (e) incubating the population of primed seeds under germination        conditions, thereby identifying plants having the targeted        insertion of the polynucleotide of interest.-   49. A method to identify a plant having a targeted insertion of a    polynucleotide of interest in its genome comprising:    -   (a) providing a population of plants having stably incorporated        into their genome a DNA construct comprising a promoter active        in a seed operably linked to a target site, the target site        comprising a first and a second recombination site, wherein the        first and the second recombination sites are dissimilar and        non-recombinogenic with each other;    -   (b) introducing into the population of plants a transfer        cassette comprising in the following order the first        recombination site operably linked to a polynucleotide encoding        a selection marker that confers resistance to a selective agent,        wherein the polynucleotide is not operably linked to a promoter,        the polynucleotide of interest, and the second recombination        site;    -   (c) providing a recombinase to the population of plants, wherein        the recombinase recognizes and implements recombination at the        first and the second recombination site;    -   (d) contacting a population of seeds produced from the        population of plants from step (c) with a priming matrix        comprising an effective concentration of the selective agent for        a time sufficient to produce a population of primed seeds; and,    -   (e) incubating the population of seeds under germination        conditions, thereby identifying plants having the targeted        insertion of the polynucleotide sequence of interest.-   50. The method of any one of 48-49, wherein introducing in step (b)    comprises sexual breeding.-   51. The method of any one of 48-50, wherein the transfer cassette    comprises in the following order: the first recombination site    operably linked to the polynucleotide encoding the selection marker    that confers resistance to the selective agent, wherein the    polynucleotide is not operably linked to a promoter; a second    promoter active in the plant operably linked to the polynucleotide    of interest; and the second recombination site.-   52. The method of any one of 48-51, wherein the transfer cassette    comprises in the following order: the first recombination site    operably linked to the polynucleotide encoding the selection marker    that confers resistance to the selective agent, wherein the    polynucleotide is not operably linked to a promoter; a second    promoter active in the plant operably linked to the polynucleotide    of interest; and a third promoter active in the plant operably    linked to the second recombination site.-   53. The method of any one of 48-52, wherein the DNA construct    comprises the promoter active in the seed operably linked to an ATG    start codon operably linked to the target site; and wherein the    transfer cassette comprises in the following order, the first    recombination site operably linked to a polynucleotide encoding a    selection marker, wherein the polynucleotide lacks an operably    linked promoter and an ATG start codon, and the polynucleotide of    interest.-   54. The method of any one of 48-53, wherein the DNA construct    comprises the promoter active in the seed operably linked to an ATG    start codon operably linked to the target site; and wherein the    transfer cassette comprises in the following order, the first    recombination site operably linked to a polynucleotide encoding a    selection marker, wherein the polynucleotide lacks an operably    linked promoter and an ATG start codon, and a second promoter active    in the plant operably linked to the polynucleotide of interest.-   55. A composition comprising a seed having stably incorporated into    its genome a DNA construct comprising, in the following order, a    promoter active in the seed operably linked to a first recombination    site operably linked to a polynucleotide encoding a selection marker    that confers resistance to a selective agent; and, a priming matrix    comprising an effective concentration of the selective agent.-   56. The composition of 55, wherein the DNA construct comprises in    the following order, the promoter operably linked to the first    recombination site operably linked to the polynucleotide encoding    the selection marker, and a second recombination site.-   57. The composition of 56, wherein the first and the second    recombination sites are dissimilar and non-recombinogenic with each    other.-   58. The composition of any one of 55-57, wherein the DNA construct    further comprises a polynucleotide of interest.-   59. The composition of any one of 55-58, wherein the DNA construct    further comprises a second selection marker.-   60. The composition of 59, wherein the second selection marker    confers resistance to a second selective agent.-   61. A method to identify a maize plant having a DNA construct stably    incorporated in its genome comprising    -   (a) providing a population of seeds, wherein at least one seed        in the population has stably incorporated into its genome the        DNA construct comprising, in the following order, a promoter        active in the seed operably linked to a first recombination site        operably linked to a polynucleotide encoding a selection marker        that confers resistance to a selective agent, and a second        recombination site;    -   (b) contacting the population of seeds with a priming matrix        comprising an effective concentration of the selective agent for        a time sufficient to produce a population of primed seeds,        wherein the selective agent comprises cyanamide and the        selection marker comprises cyanamide hydratase; and,    -   (c) incubating the population of primed seed under germination        conditions, and thereby identifying plants having the DNA        construct stably incorporated into their genome.-   62. The method of any one of 1-55, or 61, wherein the priming matrix    further comprises an additive selected from the group consisting of    glycerol, Tween-20, and NP-40.-   63. The composition of 60, wherein the priming matrix further    comprises the second selective agent.-   64. The composition of any one of 56-60 or 63, wherein the priming    matrix further comprises an additive selected from the group    consisting of glycerol, Tween-20, and NP-40.-   65. A method to identify a plant having a DNA construct stably    incorporated in its genome comprising:    -   (a) providing a population of seeds, wherein at least one seed        in the population has stably incorporated into its genome the        DNA construct comprising the following operably linked        components in the following order:        -   (i) a first expression unit comprising from 5′-3′: a first            promoter active in the seed, a first recombination site, a            first polynucleotide encoding a first selection marker that            confers resistance to a first selective agent, and a first            terminator active in a plant; and,        -   (ii) a second expression unit comprising from 5′-3′: a            second terminator active in a plant, a second polynucleotide            encoding a second selection marker that confers resistance            to a second selective agent, a second recombination site,            and a second promoter active in the seed, wherein the first            and the second selection markers confer resistance to            different selective agents;    -   (b) contacting the population of seeds with a priming matrix        comprising an effective concentration of the first and the        second selection agents for a time sufficient to produce a        population of primed seeds; and,    -   (c) incubating the population of primed seed under germination        conditions, thereby identifying plants having the DNA construct        stably incorporated into their genome.-   66. The method of 65, wherein the first and the second recombination    sites are dissimilar and non-recombinogenic with each other.-   67. The method of any one of 65-66, wherein at least one of the    first or the second selective agents comprises an herbicide, a    growth regulator, or an antibiotic.-   68. The method of any one of 65-67, wherein at least one of the    first or the second selective agents is selected from the group    consisting of glufosinate, phosphinothricin, glyphosate, bromoxynil,    methotrexate, imidazolinones, sulfonylureas, cyanamide, kanamycin,    G418, neomycin, and hygromycin B.-   69. The method of any one of 65-67, wherein at least one of the    first or the second selection markers is selected from the group    consisting of Bar, phosphinothricin acetyltransferase (PAT),    glyphosate oxidoreductase (GOX), 5-enolpyruvylshikimate-3-phosphate    synthase (EPSPS), CP4, glyphosate N-acetyltransferase (GAT),    bromoxynil nitrilase (BXN), dihydrofolate reductase, acetolactate    synthase (ALS), cyanamide hydratase (CAH), neomycin    phosphotransferase (nptII), aminoglycoside 3′-phosphotransferase    (APH3′ II), and hygromycin phosphotransferase (hph).-   70. The method of any one of 65-69, wherein either the first or the    second selective agent is cyanamide or an active derivative of    cyanamide.-   71. The method of any one of 65-70, wherein either the first or the    second selection marker is cyanamide hydratase.-   72. The method of any one of 65-71, wherein at least one of the    first or the second polynucleotide has been synthesized using plant    preferred codons.-   73. The method of 72, wherein at least one of the first or the    second polynucleotide has been synthesized using maize preferred    codons.-   74. The composition of any one of 55-60, wherein the seed is from a    plant selected from the group consisting of maize, wheat, rice,    sorghum, rye, oat, barley, millet, Brassica, alfalfa, sunflower,    safflower, soybean, tobacco, cotton, and Arabidopsis.

Compositions and methods to prime seeds are provided. For example, amethod of priming a seed comprises providing a seed having stablyincorporated into its genome a DNA construct comprising, in the 5′ to 3′or 3′ to 5′ orientation, the following operably linked components: apromoter active in the seed, a first recombination site, and apolynucleotide encoding a selection marker that confers resistance to aselective agent. This seed is contacted with a priming matrix comprisingan effective concentration of the selective agent for a time sufficientto produce a primed seed. Seeds having such a DNA construct can begenerated in a variety of ways. In some examples, the seed having such aDNA construct is a targeted seed. In some methods the DNA constructfurther comprises at least a second recombination site. In some examplesthe DNA construct further comprises a polynucleotide of interest. Insome examples the DNA construct further comprises a second selectionmarker. In some examples the first and the second recombination sitesare dissimilar and non-recombinogenic with each other. In some examplesthe seed is dried after priming. In some examples the method furthercomprises incubating the primed seed under germination conditions. Insome examples the seed priming methods and compositions provideefficient isolation and/or identification of seeds/plants having suchDNA constructs. In some examples, the priming methods allow for theidentification of a seed having an activated selectable marker.

Using traditional methods, seed expressing the selection marker would besegregated based on leaf painting and/or spraying, followed by molecularanalysis such as PCR, sequencing, ELISA, or any combination of these orsimilar techniques. The methods herein expose the seed to theappropriate selective agent during the priming stage, which inhibitsgermination in the absence of adequate resistance, providing easy, fast,and efficient screening. For example it allows a large population seedsto be screened without segregating, leaf painting, spraying, and/ormolecular analyses. Further characterization and analyses can be done,including but not limited to PCR, sequencing, Southern blots, Northernblots, Western blots, ELISA, mapping, agronomic evaluations, and thelike.

Compositions include a mixture comprising: a seed having stablyincorporated into its genome a DNA construct comprising the followingoperably linked components, a promoter active in the seed, a firstrecombination site, and a polynucleotide encoding a selection markerthat confers resistance to a selective agent; and, a priming matrixcomprising an effective concentration of the selective agent. In someexamples the DNA construct further comprises at least a secondrecombination site. In some examples the first and the secondrecombination sites are dissimilar and non-recombinogenic with eachother. In some examples the DNA construct further comprises at least onepolynucleotide of interest. In some examples the DNA construct furthercomprises a second selection marker. In some examples the priming matrixfurther comprises a second selection agent, a surfactant, a fungicide, apreservative, and/or another compound.

The methods provide for the identification of plants having stablyincorporated into their genome a DNA construct comprising in the 5′ to3′ or 3′ to 5′ orientation the following operably linked components: apromoter active in the seed, a first recombination site, and apolynucleotide encoding a selection marker that confers resistance to aselective agent. In some examples the method comprises providing apopulation of plants having stably incorporated into their genome a DNAconstruct comprising a promoter active in the seed operably linked to atarget site, the target site comprising a first recombination site;providing to the population of plants a transfer cassette comprising thefirst recombination site operably linked to a polynucleotide encoding aselection marker that confers resistance to a selective agent, whereinthe polynucleotide is not operably linked to a promoter; and, providingto the population of plants a recombinase wherein the recombinaserecognizes and implements recombination between the first recombinationsites. A population of seed can be generated from this population ofplants, this seed can be contacted with a priming matrix comprising aneffective concentration of the selective agent for a time sufficient toproduce a population of primed seed. In some examples the primed seedare then dried. In some examples the primed seed are further incubatedunder germination conditions to identify plants having the DNAconstruct. In some examples, DNA construct further comprises at least asecond recombination site. In some examples, the first and the secondrecombination sites are dissimilar and non-recombinogenic with eachother. In some examples the DNA construct further comprises apolynucleotide of interest. In some examples the polynucleotide ofinterest encodes a second selection marker. In some examples the secondselection marker confers resistance to a second selective agent. Seedshaving such a DNA construct can be generated in a variety of ways.

In some instances, the seed having such a DNA construct is a targetedseed, wherein a targeted seed or plant comprises a DNA construct thatwas generated and/or manipulated via a site-specific recombinationsystem. In one example, the generation of the targeted seed comprises:providing a population of plants having stably incorporated into theirgenome a DNA construct comprising a promoter active in the seed operablylinked to a target site, the target site comprising a firstrecombination site; providing to the population of plants a transfercassette comprising the first recombination site operably linked to apolynucleotide encoding a selection marker that confers resistance to aselective agent, wherein the polynucleotide is not operably linked to apromoter; and, providing to the population of plants a recombinasewherein the recombinase recognizes and implements recombination betweenthe first recombination sites. In some examples a population of seedfrom the population of plants is contacted with a priming matrixcomprising an effective concentration of the selective agent for a timesufficient to produce a population of primed seeds. In some examples theprimed seed is then dried. In some examples the primed seed is furtherincubated under germination conditions and plants having the targetedinsertion of the DNA construct identified. The population of plantsobtained comprises targeted plants having the transfer cassetteintegrated at the target site, and plants in which the transfer cassetteintegrated randomly in the genome. The methods and compositions provideefficient identification and/or isolation of the targeted plants. In oneexample, the transfer cassette can be provided to the plant having thetarget site by a sexual cross. In some examples the target site furthercomprises a second recombination site, wherein the first and the secondrecombination sites are dissimilar and non-recombinogenic with eachother. In some examples the transfer cassette further comprises thesecond recombination site, wherein the first and second recombinationsites are dissimilar and non-recombinogenic with each other, andcorrespond to the recombination sites in the target site, wherein arecombinase is provided which recognizes and implements recombination atthe first and second recombination sites. In some examples the targetsite comprises a first recombination site, and the transfer cassettecomprises a second recombination site, wherein the first and the secondrecombination sites are dissimilar and recombinogenic with each other.In some examples the recombinase provided comprises a biologicallyactive variant and/or fragment of the recombinase. In some examples, therecombination product of the dissimilar and recombinogenic first andsecond recombination sites comprises a modified first and modifiedsecond recombination sites which are dissimilar and non-recombinogenicwith each other, thereby inhibiting the reverse recombination reaction.The dissimilar and recombinogenic sites can have at least one nucleotidechange in any region of the site, including but not limited to arecombinase binding element, spacer region, or combination thereof. Forexample, mutant lox sites with introduced nucleotide changes into theleft 13 bp element (LE mutant lox site) or the right 13 by element (REmutant lox site) have been used (Albert et al. (1995) Plant J7:649-659). Recombination between the LE mutant lox site and the REmutant lox site produces the wild-type loxP site and a LE/RE mutant sitethat is poorly recognized by Cre, resulting in a stable integrationevent. See also, for example, Araki et al. (1997) Nucleic Acids Res25:868-872. In some examples, a recombination system is used wherein thereverse reaction is inhibited, for example, an SSV1, lambda, or phiC31integrase system using Int recombinase and appropriate att sites.

In some examples, at least one polynucleotide of interest can beincluded in a construct, for example the target site, transfer cassette,and/or DNA construct. Any polynucleotide of interest can be used,including markers, a recombinase, trait genes, regulatory sequences,insulators, operators, repressors, replication origins, binding regions,recognition sites, templates, activators, silencing constructs, and thelike. A change in phenotype may be provided by expression ofheterologous products, increased expression of endogenous products inplants, and/or reduced expression of one or more products, or anycombination thereof.

Various methods can be used to generate a targeted seed or plant. Oncethe targeted seed is generated, priming with the appropriate selectiveagent is used to identify targeted seed having the desired DNAconstruct. Methods are also provided to identify a plant having atargeted insertion of a polynucleotide of interest in its genome. Insome examples the method comprises providing a population of plantshaving stably incorporated into their genome a DNA construct comprisingin the 5′ to 3′ or 3′ to 5′ orientation a promoter active in the seedoperably linked to a target site, wherein the target site comprises afirst and a second recombination site, wherein the first and the secondrecombination sites are dissimilar and non-recombinogenic with eachother; introducing into the population of plants a transfer cassettecomprising in the 5′ to 3′ or 3′ to 5′ orientation the firstrecombination site, a polynucleotide encoding a selection marker thatconfers resistance to a selective agent, and the second recombinationsite, wherein the polynucleotide encoding the selection marker is notoperably linked to a promoter; providing to the population of plants arecombinase, wherein the recombinase recognizes and implementsrecombination at the first and the second recombination sites;contacting a population of seeds produced from the population of plantsdescribed above with a priming matrix comprising an effectiveconcentration of the selective agent for a time sufficient to produce apopulation of primed seed; and, incubating the population of primed seedunder germination conditions, thereby identifying plants having thetargeted insertion of the polynucleotide. In some examples the first andthe second recombination sites are dissimilar and recombinogenic witheach other. In some examples the recombinase provided comprises abiologically active variant and/or fragment of the recombinase.

The plants, cells, and/or seeds employed can have stably incorporatedinto their genome a DNA construct comprising in the 5′ to 3′ or 3′ to 5′orientation a promoter active in the seed operably linked to a firstrecombination site operably linked to a polynucleotide encoding aselection marker that confers resistance to a selective agent. In someexamples the DNA construct further comprises at least a secondrecombination site. In some examples, the first and the secondrecombination sites are dissimilar and non-recombinogenic with eachother. In some examples the first and the second recombination sites aredissimilar and recombinogenic with each other. In some examples the DNAconstruct further comprises a polynucleotide of interest.

Several examples of target sites, transfer cassettes, and DNA constructsinclude, but are not limited to those shown in TABLE 1. The lettersand/or numbers are for identification only, and not meant to imply anyassociation between the various constructs.

TABLE 1 Target sites A P1::RSa B P1::RSa::NT1 C P1::RSa::NT1::T1 DP1::ATG::RSa E P1::ATG::RSa::NT1(no ATG) F P1::ATG::RSa::NT1(no ATG)::T1G P1::RSa::P2 H P1::ATG::RSa::P2 I P1::RSa::ATG::P2 JP1::ATG::RSa::ATG::P2 K P1::RSa::spacer::RSb L P1::ATG::RSa::spacer::RSbM P1::RSa::NT1::T1-RSb N P1::RSa::NT1::T1-RSb::P2 O P1::ATG::RSa::NT1(noATG)::T1-RSb::P2 P P1::RSa::NT1::T1-RSb::P2::NT2::T2 QP1::ATG::RSa::NT1(no ATG)::T1-RSb::P2::NT2::T2 RP1::RSa::NT1::T1-P2::NT2::T2-RSb S P1::RSa::NT1::T1-P2::NT2::T2-P3::RSbT P1::ATG::RSa::NT1(no ATG)::T1- P2::NT1::T2::RSb U P1::ATG::RSa::NT1(noATG)::T1- P2::NT1::T3- P3::ATG::RSb VP1::RSa::NT1::T1-P2::NT2::T2-P3::RSb::S2::T3- P4::NT3::T4::RSc WP1::ATG::RSa::NT1(no ATG)::T1- P2::NT2::T2-P3::RSc Transfer Cassettes  1RSa::S1  2 RSa::S1::T1  3 RSa::S1(no ATG)  4 RSa::S1(no ATG)::T1  5T2::S2::RSa::S1::T1 (circular)  6 T2::S2::RSa::S1(no ATG)::T1 (circular) 7 T2::S2(no ATG)::RSa::S1::T1 (circular)  8 T2::S2(no ATG)::RSa::S1(noATG)::T1  9 RSa::S1::T1-P2::NT1 10 RSa::S1::T1-P2::NT1::T2 11 RSa::S1(noATG)::T1-P2::NT1 12 T3::S2::RSa::S1::T1-P2::NT1::T2 (circular) 13RSa::S1-RSb 14 RSa::S1::T1-RSb 15 RSa::S1::T1-P2::NT1::T2-RSb 16RSa::S1::T1-P2::NT1::T2-P3::RSb 17 RSa::S1(no ATG)::T1- P2::NT1::T2::RSb18 RSa::S1::T1- P2::NT1::T3- P3::ATG::RSb 19 RSa::S1(no ATG)::T1-P2::NT1::T3- P3::ATG::RSb 20RSa::S1::T1-P2::NT1::T2-P3::RSb::S2::T3-P4::NT2::T4::RSc 21RSa::S1::T1-P2::NT1::T2-P3::RSc::S2::T3-P4::NT2::T4::RSb 22 RSa::S1(noATG)::T1- P2::NT1::T2-P3::ATG::RSb::S2(no ATG)::T3-P4::NT2::T4::RSc DNAconstructs a P1::RSa::S1 b P1::RSa::S1::T1 c P1::ATG::RSa::S1(no ATG) dP1::ATG::RSa::S1(no ATG)::T1 e P1::RSa::S1::T1-T2::S2::RSa::P2 fP1::ATG::RSa::S1(no ATG)::T1-T2::S2::RSa::P2 g P1::RSa::S1::T1-T2::S2(noATG)::RSa::ATG::P2 h P1::ATG::RSa::S1(no ATG)::T1-T2::S2(noATG)::RSa::ATG::P2 i P1::RSa::S1::T1-P2::NT1 jP1::RSa::S1::T1-P2::NT1::T2 k P1::ATG::RSa::S1(no ATG)::T1-P2::NT1 lP1::RSa::S1::T1-P2::NT1::T2-T3::S2::RSa::P3 m P1::RSa::S1-RSb nP1::RSa::S1::T1-RSb o P1::RSa::S1::T1-P2::NT1::T2-RSb pP1::RSa::S1::T1-P2::NT1::T2-P3::RSb q P1::ATG::RSa::S1(no ATG)::T1-P2::NT1::T2::RSb r P1::RSa::S1::T1- P2::NT1::T3- P3::ATG::RSb sP1::ATG::RSa::S1(no ATG)::T1- P2::NT1::T3- P3::ATG::RSb tP1::RSa::S1::T1-P2::NT1::T2-P3::RSb::S2::T3- P4::NT2::T4::RSc uP1::ATG::RSa::S1(no ATG)::T1- P2::NT1::T2- P3::ATG::RSb::S2(no ATG)::T3-P4::NT2::T4::RSc TABLE 1: P = promoter; RS = recombination site; S =selection marker; T = terminator region; NT = polynucleotide ofinterest; ATG = start codon; no ATG indicates that the coding regionlacks the ATG start codon; the symbol :: indicates an operable linkageor fusion between adjacent elements; and ATG::RS indicates an operablein-frame fusion to produce a properly expressed functional gene product.

In some examples the plant comprises a DNA construct comprising a firstexpression unit comprising the following operably linked components in5′ to 3′ or 3′ to 5′ orientation: a promoter active in a seed, a firstrecombination site, and a selection marker. In some examples, atermination region is operably linked to the selection marker. In someexamples the DNA construct further comprises a second promoter. In someexamples, a polynucleotide of interest is operably linked to the secondpromoter, optionally with a termination region. In some examples, theDNA construct can further comprise at least a second recombination site.In some examples, the first and the second recombination sites areidentical. In further examples, the first and the second recombinationsites are dissimilar and non-recombinogenic with each other.

In some examples, the plant comprises a DNA construct comprising a firstexpression unit comprising the following operably linked components in5′ to 3′ or 3′ to 5′ orientation: promoter active in a seed, an ATGcodon, a first recombination site, and a selection marker, wherein theselection marker lacks an ATG start codon (P1::ATG::RSa::S1(no ATG)). Insome examples, a termination region is operably linked to the selectionmarker. In some examples the DNA construct further comprises a secondpromoter. In some examples, a polynucleotide of interest is operablylinked to the second promoter. Optionally the polynucleotide of interestis operably linked to a termination region. In some examples, the DNAconstruct further comprises at least a second recombination site. Insome examples, the first and the second recombination sites areidentical. In further examples, the first and the second recombinationsites are dissimilar and non-recombinogenic with each other.

In some examples, the plant comprises a DNA construct comprising a firstand a second expression unit, wherein the first expression unitcomprises following operably linked components in 5′ to 3′ or 3′ to 5′orientation: a promoter active in a seed, a first recombination site,and a first selection marker, optionally linked to a termination region,and the second expression cassette comprises the following operablylinked components in 5′ to 3′ or 3′ to 5′ orientation: a terminationregion, a second selection marker, a second recombination site, and asecond promoter. In some examples, the first and the secondrecombination sites are identical. In further examples, the first andthe second recombination sites are dissimilar and non-recombinogenicwith each other. In some examples, at least one promoter is operablylinked to an ATG, wherein the operably linked selection marker lacks anATG start codon. In some examples the DNA construct further comprises apromoter operably linked to a polynucleotide of interest.

In some instances, the plant comprises a DNA construct comprising afirst and a second expression unit, where the first expression unitcomprises a first promoter active in a seed operably linked to a firstrecombination site operably linked to a selection marker. The secondexpression unit comprises a second promoter active in the plant operablylinked to a polynucleotide of interest operably linked to a secondrecombination site. In some examples, the first and the secondrecombination sites are dissimilar and non-recombinogenic with eachother. Optionally, at least one terminator region may be operably linkedto the first and/or second expression unit.

In other instances, the DNA construct comprises a first, a second, and athird expression unit. The first expression unit comprises the followingoperably linked components in 5′ to 3′ or 3′ to 5′ orientation: apromoter active in a seed, a first recombination site, and a selectionmarker. The second expression unit comprises a second promoter active inthe plant operably linked to a polynucleotide of interest. The thirdexpression cassette comprises a third promoter active in the plantoperably linked to a second recombination site. In some examples, thefirst and the second recombination sites are dissimilar andnon-recombinogenic with each other. Optionally, at least one terminatorregion may be operably linked to the first, second, and/or thirdexpression unit.

In other examples, the DNA construct comprises a first and a secondexpression unit. The first expression unit comprises the followingoperably linked components in 5′ to 3′ or 3′ to 5′ orientation: apromoter active in the seed, an ATG start codon, a first recombinationsite, and a selection marker, wherein the selection marker lacks an ATGstart codon. The second expression unit comprises a second promoteractive in the plant operably linked to a polynucleotide of interest, anda second recombination site. In some cases, the first and the secondrecombination sites are dissimilar and non-recombinogenic with eachother. Optionally, at least one terminator region may be operably linkedto the first and/or second expression unit.

In other examples, the DNA construct comprises a first, a second, athird, and a fourth expression unit. The first expression unit comprisesthe following operably linked components in 5′ to 3′ or 3′ to 5′orientation: a promoter active in the seed, an ATG start codon, a firstrecombination site, and a selection marker, wherein the selection markerlacks an ATG start codon. The second expression unit comprises a secondpromoter active in a plant operably linked to a polynucleotide ofinterest. The third expression unit comprises the following operablylinked components in 5′ to 3′ or 3′ to 5′ orientation: a third promoteractive in a plant, an ATG start codon, a second recombination site, anda second selection marker, wherein the second selection marker lacks anATG start codon. The fourth expression unit comprises the followingoperably linked components in 5′ to 3′ or 3′ to 5′ orientation: a fourthpromoter active in the plant, a second polynucleotide of interest, and athird recombination site. In some examples, the first, the second,and/or the third recombination sites are dissimilar andnon-recombinogenic with each other. Optionally, at least one terminatorregion may be operably linked to the first, second, third and/or fourthexpression unit.

In other examples, an expression unit can comprise a promoter operablylinked to a first polynucleotide and a second polynucleotide such thatthe single promoter drives expression of both polynucleotides.Optionally, at least one terminator region may be operably linked to thefirst, and/or second polynucleotide sequence.

In some examples, target sites and transfer cassettes are used tomanipulate, exchange, excise, invert, alter, and/or introduce the DNAconstruct. In some examples, the recombination sites are dissimilar andnon-recombinogenic with each other. In some examples, the recombinationsites are dissimilar and recombinogenic with each other. One or moreintervening sequences may be present between the recombination sites ofthe target site and/or transfer cassette. In some examples, therecombination sites of the target site and/or transfer cassette may bedirectly contiguous with the selection marker and/or a polynucleotide ofinterest. Intervening sequences of interest include linkers, adapters,selectable markers, additional polynucleotides of interest, promoters,regulatory sequences, insulators, binding regions, recognition sites,repressors, operators, activators and/or other sites/sequences that aidin vector construction, expression, or analysis. In addition, therecombination site(s) of the target site can be located and/or orientedin various positions, including, for example, within intronic sequences,coding sequences, or untranslated regions. In certain examples, therecombination sites can be contained within the selectable marker and/orthe polynucleotide of interest, for example within an intron, codingsequence, or untranslated region. In some examples, the plant maycomprise multiple target sites at one or more locations in the genome.When more than one target site is at one locus in the genome, multiplemanipulations of this target locus in the plant are available. In someexamples, the genome of the plant having the target site may alsocomprise an expression cassette comprising a polynucleotide encoding anappropriate recombinase. In another example, the target site itselfcomprises a nucleotide sequence encoding a recombinase. Once a targetsite has been established within a genome it is possible to subsequentlyadd, remove, and/or alter sites through recombination, for example,additional recombination sites may be introduced by incorporating suchsites within the nucleotide sequence of the transfer cassette (see, e.g.WO99/25821).

Multiple genes or polynucleotides of interest can be stacked, ordered,and/or manipulated at a precise genomic location in the plant. Forexample, the DNA construct can comprise the following components:P1::RSa::S1::T1-P2::NT1::T2-P3::RSb-RSc. In certain examples, P3::RSballows priming/selection to be repeated by introducing a differentsecond selection marker. For example, once the construct is incorporatedinto the genome, a transfer cassette comprising the following componentscould be introduced: RSb::S2::T3-P4::NT2::T4-RSc. The priming methodscan then be used to select seed expressing the second selection marker,or the first and the second marker. In this manner, multiple sequencescan be stacked at predetermined locations. Various alterations can bemade to stack the polynucleotides of interest in the genome of theplant. The targeted seeds produced can have a simple integration patterncomprising integration exclusively or predominantly at the target sitewith no or very few random insertions elsewhere in the genome. Methodsfor determining the integration patterns are known and include, forexample, Southern blot analysis and RFLP analysis.

The activity of various promoters at a characterized location in theplant genome can be determined, and compared. Further, the activity,expression pattern, and/or expression level of a polynucleotide ofinterest can be determined. For example, a method for assessing promoteractivity in a plant comprises introducing into the plant a transfercassette comprising a first recombination site operably linked to aselection marker, wherein the selection marker is not operably linked toa promoter, a second promoter active in the plant operably linked to apolynucleotide of interest and the second recombination site. The plantfurther comprises in its genome a DNA construct comprising a firstpromoter active in the seed operably linked to a target site comprisingthe first and the second recombination site, wherein the first and thesecond recombination sites are dissimilar and non-recombinogenic witheach other. A recombinase is provided, wherein the recombinaserecognizes and implements recombination at the first and the secondrecombination sites. Seeds are then primed with an appropriate selectiveagent to identify targeted seeds wherein the transfer cassette hasintegrated at the target site. Such targeted seeds, or plants derivedtherefrom, can further be tested to assess the promoter activity of thesecond promoter. Once the activity of the promoter is characterized,additional transfer cassettes can be employed to allow the characterizedpromoter to drive expression of the polynucleotide of interest. In someinstances, the activity of the second promoter can further be comparedto the activity of the first promoter. Plant lines having suchcharacterized promoters can be engineered so that polynucleotides ofinterest can be operably linked to the promoter, and thereby expressedin a desired manner. In some examples the recombinase provided comprisesa biologically active variant and/or fragment of the recombinase.

The DNA constructs, transfer cassettes, and/or target sites can bedesigned to minimize or eliminate expression resulting from randomintegration of DNA sequences into the genome of a plant, for example,the transfer cassettes can be designed without either of a promoter oran ATG start codon operably linked to the selection marker. The targetsite comprises a promoter operably linked to a recombination site whichis further operably linked to an ATG translation start site. Randomintegration of the transfer cassette is unlikely to produce expressionof the selection marker, since the transfer cassette would need torandomly integrate behind an endogenous promoter region, an ATG startsite, and in the correct reading frame.

A plant is provided having stably incorporated into its genome a DNAconstruct comprising the following operably linked components in thefollowing order: a first promoter active in the seed, an ATG startcodon, and a target site comprising a first recombination site. Atransfer cassette is introduced into the plant comprising, in thefollowing order: the first recombination site operably linked to apolynucleotide encoding a selection marker, wherein the polynucleotideencoding the selectable marker lacks an ATG start codon, and thepolynucleotide encoding the selectable marker not operably linked to apromoter. A recombinase is provided that recognizes and implementsrecombination between the first recombination sites, which results inthe selection marker being operably linked to the first promoter and theATG start codon of the target site. Expression of the selection markeris controlled by the first promoter, therefore priming with theappropriate selective agent can be used to identify the targeted seed.In some examples the target site and/or the transfer cassette furthercomprises at least a second recombination site. In some examples thefirst and the second recombination sites are dissimilar andnon-recombinogenic with each other. In some examples the first and thesecond recombination sites are dissimilar and recombinogenic with eachother. In some examples the transfer cassette further comprises a secondpromoter operably linked to a polynucleotide of interest. In someexamples, plants comprising the target site are crossed with plantscomprising the transfer cassette to generate seeds/plants comprising theDNA construct. In some examples the recombinase comprises a biologicallyactive variant and/or fragment of the recombinase.

Seed dormancy is a unique form of developmental arrest utilized by mostplants to temporally delay germination and optimize progeny survival.During seed dormancy, moisture content and respiration rate aredramatically lowered. The initial step to break seed dormancy is theuptake (imbibition) of water to increase respiration and mobilization ofstarch reserves required for germination. The water uptake is triphasic,including an initial rapid period (phase I), followed by apre-germination plateau (phase II) in which water uptake is slower andless intense than in phase I. In phase III, there is a subsequentincrease in water uptake which results in the growth of the embryonicaxis, radicle emergence, and resumption of growth, i.e., germination.The three phases are separated temporally, and a seed that has enteredand/or completed phase I and II is said to be a primed seed, that is,primed for phase III: germination. Seed priming enables faster and moreuniform germination upon sowing or planting of seed. Priming providesthe option of simultaneously treating the seed with fungicide,preservatives, or other agents, to provide protection during processing,after sowing, and/or to prolong viable storage time. Seed priming allowsthe seeds to begin the pre-germinative metabolic processes, and thenarrests the seeds at the pre-germinative stage. The amount of waterabsorbed is carefully controlled to avoid germination or seed ageing.Once the seed has absorbed sufficient water, the seed can be held atthat water content, or dried back to the original water content forstorage. Primed seeds can be sown directly without drying whereupon theygerminate faster than seeds which have been primed and dried.

A priming matrix is a composition comprising an effective concentrationof a selective agent and having an effective osmotic potential. Aneffective concentration of a selective agent is sufficient to prevent orsignificantly reduce the germination of seed that do not express thecorresponding selection marker. An effective concentration of aselective agent comprises a concentration that prevents the germinationof at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% of a population of seeds that do not express thecorresponding selection marker. An effective osmotic potential is anosmotic potential that lowers the water potential allowing or causingwater to move into the seed to a level sufficient to prime the seed.Seeds germinate when water potential reaches a critical physiologicallevel which varies between plant species, but typically falls between 0and −2 Mpa. Many priming matrices that provide an appropriate osmoticpotential are available, including water, water with at least onesolute, solid matrices, and the like. For example, the priming matrixmay comprise an aerated solution of osmotic material, such aspolyethylene glycol (PEG) (see U.S. Pat. No. 5,119,598), glycerol,mannitol, and the like. Alternatively, seeds may be primed using a solidmatrix. A solid matrix material should have a high water holdingcapacity to allow seeds to imbibe. In this method, the priming matrixcan comprise an absorbent medium such as clay, vermiculite, perlite, sawdust, corn cobs, and/or peat to absorb water and then transfer it to theseed (see U.S. Pat. No. 4,912,874). The extent of hydration iscontrolled by altering the water content of the medium and themedium/seed ratio. Methods are also known to imbibe seeds in a slurry ofPEG 6000 and vermiculite, or other matrices (Peterson (1976) Sci Hort5:207-214; and U.S. Pat. No. 5,628,144). In still other methods, primingemploys a semi-permeable membrane that mediates the transfer of waterfrom a solution of set osmotic pressure to the seed (see, U.S. Pat. No.5,873,197). In other methods, ultrasonic energy can be used to assist inthe priming process (see, e.g., U.S. Pat. No. 6,453,609). Optionally avariety of additives, chemicals, and/or compounds can be included in thepriming matrix, including surfactants, additional selective agents,fungicides, agents to modify osmotic potential, osmotic protectants,agents to aid drying or protect the seed during drying, agents toenhance seed processing, agents to extend storage shelf life, agents toenhance coating and/or perfusion, agents to enhance germination of theseed, and the like. Fungicides can be included in the priming matrix,for example, thiram, captan, metalaxyl, pentachloronitrobenzene,fenaminosulf, bactericides or other preservatives. In addition, variousgrowth regulators/hormones, such as gibberellins/gibberellic acid,cytokinins, inhibitors of abscissic acid, 2-(3,4-dichlorophenoxy)triethylamine (DCPTA), potassium nitrate, and ethephon can also bepresent in the priming matrix. Other optional agents include glycerol,polyethylene glycol, mannitol, DMSO, Triton X-100, Tween-20, NP-40,ionic compounds, non-ionic compounds, surfactants, detergents, and thelike. A time sufficient to produce a primed seed allows pre-germinativemetabolic processes to take place within the seed up to any levelincluding that immediately preceding radicle-emergence. The time toproduce a primed seed is dependent on the specific seed variety, itsstate or condition, and the water potential of the priming matrix. Whiletypical water amounts and media water potentials for given seed typesare already generally known for some seeds, it is frequently best totest a small sample of a new seed over a readily determined range ofosmotic potentials and temperatures to determine what conditions oftemperature, water potential, and time provide appropriate imbibing ofthe seed and resultant pre-germination events. The temperature at whichthe priming methods are carried out may vary with the seeds to betreated, but typically is between 18° C. to 30° C. The primed seeds maybe retained in the priming matrix through germination as denoted byradical emergence. Seed produced by this method may be further dried asin U.S. Pat. No. 4,905,411.

Methods to determine if a seed has been primed are known. For example,optimization of priming treatments can be performed by carrying outgermination assays. See, for example, Jeller et al. (2003) Braz J Biol63:61-68. In addition, molecular markers of germination and/or primingare known. See, for example, Job et al. (2000) Seed Biology: Advancesand Applications, Eds. Black et al., CABI International, Wallingford,UK, pp. 449-459; De Castro et al. (2000) Plant Physiol 122:327-335;Bradford et al. (2000) Seed Biology: Advances and Applications, Eds.Black et al., CABI International, Wallingford, UK pp. 221-251; andGallardo et al. (2001) Plant Physiol 126:835-848.

After priming, the seeds may be allowed to germinate, or the primedseeds can be dried. The appropriate conditions (temperature, relativehumidity, and time) for the drying process will vary depending on theseed and can be determined empirically. See, for example, Jeller et al.(2003) Braz J Biol 63:61-68. Drying primed seed includes a superficialdrying of the seed or, alternatively, drying the seed back to itsoriginal water content. The dried seeds can be immediately germinated orcan be stored under appropriate conditions. Germination conditions forvarious seed are known. One factor in determining appropriategermination conditions is the threshold germination temperature range,which is the range of temperatures for a species within which seeds ofthat species will germinate at a predetermined moisture level and withadequate oxygen. Another factor is the threshold germination moisturerange, which is the range of moisture for a species within which seedsof the species will germinate at a given temperature and with adequateoxygen. Threshold germination temperature range and/or thresholdgermination moisture range values are known for various seeds, as aremethods to empirically determine these conditions for any given seed andvariety.

In some examples, the population of primed seeds is incubated undergermination conditions. Primed seed expressing the selection maker areresistant to the selective agent and will germinate to produce a viableplant. Primed seeds not expressing the selection marker will not beresistant to the selection agent and will not germinate, or germinate ata significantly lower frequency, or will germinate only to produce acoleoptile and then die, or will germinate with an abnormal phenotypeand typically die prior to the V2 stage of development. Plantsexpressing the selection marker will thereby be identified and/orselected.

The methods and compositions further employ elements from recombinationsystems, such as recombinases and recombination sites, for example in aDNA construct, a target site, and/or a transfer cassette. A target sitecomprises a polynucleotide integrated into the genome comprising apromoter operably linked to at least one recombination site. A transfercassette comprises at least a first recombination site operably linkedto a polynucleotide encoding a selection marker, and optionally apolynucleotide of interest, wherein the first recombination site isrecombinogenic with a recombination site in the target site. A targetedseed or plant has stably incorporated into its genome a DNA constructthat has been generated and/or manipulated through the use of arecombination system. Site-specific recombination methods that result invarious integration, alteration, exchange, replacement, and/or excisionevents to generate the recited DNA construct can be employed to generatea targeted seed. See, e.g., WO99/25821, WO99/25854, WO99/25840,WO99/25855, WO99/25853, WO99/23202, WO99/55851, WO01/07572, WO02/08409,and WO03/08045.

A recombinase is a polypeptide that catalyzes site-specificrecombination between its compatible recombination sites, and includesnaturally occurring recombinase sequences, variants, and/or fragmentsthat retain activity. A recombination site is a nucleotide sequence thatis specifically recognized by a recombinase enzyme, and encompassesnaturally occurring recombination site sequences, variants, and/orfragments that retain activity. For reviews of site-specificrecombinases, see Sauer (1994) Curr Op Biotech 5:521-527; Sadowski(1993) FASEB 7:760-767; Groth & Calos (2004) J Mol Biol 335:667-678; andSmith & Thorpe (2002) Mol Microbiol 44:299-307. Any recombinationsystem, or combination of systems, can be used including but not limitedto recombinases and recombination sites from the Integrase and Resolvasefamilies, biologically active variants and fragments thereof, and/or anyother naturally occurring or recombinantly produced enzyme or variantthereof that catalyzes conservative site-specific recombination betweenspecified recombination sites, and naturally occurring or modifiedrecombination sites or variants thereof that are specifically recognizedby a recombinase to generate a recombination event.

The recombination sites employed can be corresponding sites ordissimilar sites. Corresponding recombination sites, or a set ofcorresponding recombination sites, are sites having an identicalnucleotide sequence. A set of corresponding recombination sites, in thepresence of the appropriate recombinase, will efficiently recombine withone another. Dissimilar recombination sites have a distinct sequence,comprising at least one nucleotide difference as compared to each other.The recombination sites within a set of dissimilar recombination sitescan be either recombinogenic or non-recombinogenic with respect to oneother. Each recombination site within the set of dissimilar sites isbiologically active and can recombine with an identical site.Recombinogenic sites are capable of recombining with one another in thepresence of the appropriate recombinase. Recombinogenic sites includethose sites where the relative excision efficiency of recombinationbetween the recombinogenic sites is above the detectable limit understandard conditions in an excision assay as compared to the wild typecontrol, typically, greater than 2%, 5%, 10%, 20%, 50%, 100%, orgreater. Non-recombinogenic sites will not recombine with one another inthe presence of the appropriate recombinase, or recombination betweenthe sites is not detectable. Non-recombinogenic recombination sitesinclude those sites that recombine with one another at a frequency lowerthan the detectable limit under standard conditions in an excision assayas compared to the wild type control, typically, lower than 2%, 1.5%,1%, 0.75%, 0.5%, 0.25%, 0.1%, 0.075, 0.005%, or 0.001%. Any suitablenon-recombinogenic recombination sites may be utilized, including a FRTsite or active variant thereof, a lox site or active variant thereof, anatt site or active variant thereof, any combination thereof, or anyother combination of non-recombinogenic recombination sites. Directlyrepeated recombination sites in a set of recombinogenic recombinationsites are arranged in the same orientation, recombination between thesesites results in excision of the intervening DNA sequence. Invertedrecombination sites in a set of recombinogenic recombination sites arearranged in the opposite orientation, recombination between these sitesresults in inversion of the intervening DNA sequence.

The Integrase family of recombinases has over one hundred members andincludes, for example, FLP, Cre, Int, and R. For other members of theIntegrase family, see for example, Esposito et al. (1997) Nucleic AcidsRes 25:3605-3614; Nunes-Duby et al. (1998) Nucleic Acids Res 26:391-406;Abremski et al. (1992) Protein Eng 5:87-91; Groth & Calos (2004) J MolBiol 335:667-678; and Smith & Thorpe (2002) Mol Microbiol 44:299-307.Other recombination systems include, for example, streptomycetebacteriophage phiC31 (Kuhstoss et al. (1991) J Mol Biol 20:897-908);bacteriophage λ (Landy (1989) Ann Rev Biochem 58:913-949, and Landy(1993) Curr Op Genet Dev 3:699-707); SSV1 site-specific recombinationsystem from Sulfolobus shibatae (Maskhelishvili et al. (1993) Mol GenGenet 237:334-342); and a retroviral integrase-based integration system(Tanaka et al. (1998) Gene 17:67-76). In some examples, the recombinaseis one that does not require cofactors or a supercoiled substrate. Suchrecombinases include Cre, FLP, phiC31 Int, mutant λ Int, or activevariants or fragments thereof. FLP recombinase catalyzes a site-specificreaction between two FRT sites, and is involved in amplifying the copynumber of the two-micron plasmid of S. cerevisiae during DNAreplication. The FLP protein has been cloned and expressed. See, forexample, Cox (1993) Proc Natl Acad Sci USA 80:4223-4227. The FLPrecombinase used may be derived from the genus Saccharomyces. In someexamples a polynucleotide encoding the recombinase synthesized usingplant-preferred codons is used. FLP enzyme encoded by a polynucleotidecomprising maize preferred codons (FLPm) that catalyzes site-specificrecombination events is known (U.S. Pat. No. 5,929,301). Additionalfunctional variants and fragments of FLP are known. See, for example,Buchholz et al. (1998) Nat Biotechnol 16:617-618, Hartung et al. (1998)J Biol Chem 273:22884-22891, Saxena et al. (1997) Biochim Biophys Acta1340:187-204, Hartley et al. (1980) Nature 286:860-864, Shaikh &Sadowski (2000) J Mol Biol 302:27-48, Voziyanov et al. (2002) NucleicAcids Res 30:1656-1663, and Voziyanov et al. (2003) J Mol Biol326:65-76. The bacteriophage P1 recombinase Cre catalyzes site-specificrecombination between two lox sites. See, for example, Guo et al. (1997)Nature 389:40-46; Abremski et al. (1984) J Biol Chem 259:1509-1514; Chenet al. (1996) Somat Cell Mol Genet 22:477-488; Shaikh et al. (1977) JBiol Chem 272:5695-5702; and Buchholz et al. (1998) Nat Biotechnol16:617-618. Cre polynucleotide sequences may also be synthesized usingplant-preferred codons, for example, moCre (see, e.g., WO 99/25840), andother variants are known, see for example Vergunst et al. (2000) Science290:979-982, Santoro & Schulz (2002) Proc Natl Acad Sci USA99:4185-4190, Shaikh & Sadowski (2000) J Mol Biol 302:27-48, Rufer &Sauer (2002) Nucleic Acids Res 30:2764-2771, Wierzbicki et al. (1987)Mol Biol 195:785-794, Petyuk et al. (2004) J Biol Chem 279:37040-37048,Hartung & Kisters-Wolke (1998) J Biol Chem 273:22884-22891, Koresawa etal. (2000) J Biochem (Tokyo) 127:367-372, U.S. Pat. No. 6,890,726, andBuchholz & Stewart (2001) Nat Biotechnol 19:1047-1052. The phiC31integrase and variants are known (Kushtoss et al. (1991) J Mol Biol222:897-908, WO03/066867, WO05/017170, US2005/0003540, and Sclimenti etal. (2001) Nucleic Acids Res 29:5044-5051). The λ integrase andcofactors (Hoess et al. (1980) Proc Natl Acad Sci USA 77:2482-2486,Blattner et al. (1997) Science 277:1453-1474), and variants thereof areknown, including cofactor-independent Int variants (Miller et al. (1980)Cell 20:721-729, Lange-Gustafson and Nash (1984) J Biol Chem259:12724-12732, Christ et al. (1998) J Mol Biol 288:825-836, andLorbach et al. (2000) J Mol Biol 296:1175-1181), att site recognitionvariants (Dorgai et al. (1995) J Mol Biol 252:178-188, Yagu et al.(1995) J Mol Biol 252:163-167, and Dorgai et al. (1998) J Mol Biol277:1059-1070), as well as maize codon optimized Int, variant, andcofactor sequences (WO03/08045). Other integrases and variants areknown, such as HK022 integrase (Kolot et al. (1999) Mol Biol Rep26:207-213) and variants such as att site recognition variants (Dorgaiet al. (1995) J Mol Biol 252:178-188, Yagu et al. (1995) J Mol Biol252:163-167, and Dorgai et al. (1998) J Mol Biol 277:1059-1070).

Wild-type recombination sites, mutant sites, or any combination of wildtype and/or mutant sites can be used. Mutant sites refer to anybiologically active variant or modification of a wild type siteincluding nucleotide substitution variants in any region, and fragmentsof a full-length wild type site. Such recombination sites include, forexample, wild type lox, FRT, and att sites, and mutant lox, FRT, and attsites. An analysis of the recombination activity of mutant lox sites ispresented in Lee et al. (1998) Gene 216:55-65. Other recombination sitesand variants are known, see for example, Hoess et al. (1982) Proc NatlAcad Sci USA 79:3398-3402; Hoess et al. (1986) Nucleic Acids Res14:2287-2300; Thomson et al. (2003) Genesis 36:162-167; Schlake & Bode(1994) Biochemistry 33:12746-12751; Siebler & Bode (1997) Biochemistry36:1740-1747; Huang et al. (1991) Nucleic Acids Res 19:443-448; Sadowski(1995) in Progress in Nucleic Acid Research and Molecular Biology Vol.51, pp. 53-91; Cox (1989) in Mobile DNA, Berg & Howe (eds) AmericanSociety of Microbiology, Washington D.C., pp. 116-670; Dixon et al.(1995) Mol Microbiol 18:449-458; Umlauf & Cox (1988) EMBO J 7:1845-1852;Buchholz et al. (1996) Nucleic Acids Res 24:3118-3119; Kilby et al.(1993) Trends Genet 9:413-421; Rossant & Geagy (1995) Nat Med 1:592-594;Bayley et al. (1992) Plant Mol Biol 18:353-361; Odell et al. (1990) MolGen Genet 223:369-378; Dale & Ow (1991) Proc Natl Acad Sci USA88:10558-10562; Qui et al. (1994) Proc Natl Acad Sci USA 91:1706-1710;Stuurman et al. (1996) Plant Mol Biol 32:901-913; Dale et al. (1990)Gene 91:79-85; Albert et al. (1995) Plant J 7:649-659, U.S. Pat. No.6,465,254, WO01/23545, WO99/55851, and WO01/11058. In some examples,sets of dissimilar and corresponding recombination sites can be used,for example, sites from different recombination systems such as a setcomprising a wild type FRT site and wild type loxP site. Accordingly,any suitable recombination site or set of recombination sites may beused, including a FRT site, a biologically active variant of a FRT site,a lox site, a biologically active variant of a lox site, an att site, abiologically active variant of an att site, any combination thereof, orany other combination of recombination sites. Examples of FRT sitesinclude, for example, the minimal wild type FRT site (FRT1), and variousmutant FRT sites, including but not limited to FRT5, FRT6, and FRT7 (seeU.S. Pat. No. 6,187,994). Additional variant FRT sites are known, (see,e.g., WO 01/23545, and U.S. Provisional Ser. No. 60/700,225, filed Jul.18, 2005, herein incorporated by reference). Other recombination sitesthat can be used include att sites and variants, such as those disclosedin Landy (1989) Ann Rev Biochem 58:913-949, Landy (1993) Curr Op GenetDev 3:699-707, U.S. Pat. No. 5,888,732, WO01/07572, and Thygarajan etal. (2001) Mol Cell Biol 21:3926-3934; and lox sites and variants (see,e.g., Albert et al. 1995 Plant J 7:649-659, Hoess et al. 1986 Nucl AcidsRes 14:2287-2300, Lee & Saito 1998 Gene 216:55-65, U.S. Pat. No.6,465,254, and WO01/11058). The site-specific recombinase(s) used dependon the recombination sites in the target site and the transfer cassette.If FRT sites are utilized, FLP recombinase is provided, when lox sitesare utilized, Cre recombinase is provided, when λ att sites are used, λInt is provided, when phiC31 att sites are used, phiC31 Int is provided.If the recombination sites used comprise sites from different systems,for example a FRT and a lox site, both recombinase activities can beprovided, either as separate entities, or as a chimeric recombinase, forexample FLP/Cre (see, e.g., WO 99/25840).

A marker provides for the identification and/or selection of a cell,plant, and/or seed expressing the marker. Markers include, e.g.,screenable, visual, and/or selectable markers. A selection marker is anymarker, which when expressed at a sufficient level, confers resistanceto a selective agent. Selection markers and their correspondingselective agents include, but are not limited to, herbicide resistancegenes and herbicides; antibiotic resistance genes and antibiotics; andother chemical resistance genes with their corresponding chemicalagents. Bacterial drug resistance genes include, but are not limited to,neomycin phosphotransferase II (nptII) which confers resistance tokanamycin, paromycin, neomycin, and G418, and hygromycinphosphotransferase (hph) which confers resistance to hygromycin B. Seealso, Bowen (1993) Markers for Plant Gene Transfer, Transgenic Plants,Vol. 1, Engineering and Utilization; Everett et al. (1987)Bio/Technology 5:1201-1204; Bidney et al. (1992) Plant Mol Biol18:301-313; and WO97/05829. Resistance may also be conferred toherbicides from several groups, including amino acid synthesisinhibitors, photosynthesis inhibitors, lipid inhibitors, growthregulators, cell membrane disrupters, pigment inhibitors, seedlinggrowth inhibitors, including but not limited to imidazolinones,sulfonylureas, triazolopyrimidines, glyphosate, sethoxydim, fenoxaprop,glufosinate, phosphinothricin, triazines, bromoxynil, and the like. See,for example, Holt (1993) Ann Rev Plant Physiol Plant Mol Biol44:203-229; and Miki et al. (2004) J Biotechnol 107:193-232. Selectionmarkers include sequences that confer resistance to such herbicides andinclude, but are not limited to, the bar gene, which encodesphosphinothricin acetyl transferase (PAT) which confers resistance toglufosinate (Thompson et al. (1987) EMBO J 6:2519-2523); glyphosateoxidoreductase (GOX), glyphosate N-acetyltransferase (GAT), and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) each of which confersresistance to glyphosate (Barry et al. (1992) in Biosynthesis andMolecular Regulation of Amino Acids in Plants, B. K. Singh et al. (Eds)pp. 139-145; Kishore et al. (1992) Weed Tech 6:626-634; Castle (2004)Science 304:1151-1154; Zhou et al. (1995) Plant Cell Rep 15:159-163;WO97/04103; WO02/36782; and WO03/092360). Other selection markersinclude dihydrofolate reductase (DHFR), which confers resistance tomethotrexate (see, e.g., Dhir et al. (1994) Improvements of CerealQuality by Genetic Engineering, R. J. Henry (ed), Plenum Press, NewYork; and Hauptmann et al. (1988) Plant Physiol 86:602-606).Acetohydroxy acid synthase (AHAS or ALS) mutant sequences lead toresistance to imidiazolinones and/or sulfonylureas such as imazethapyrand/or chlorsulfuron (see, e.g., Zu et al. (2000) Nat Biotechnol18:555-558; U.S. Pat. Nos. 6,444,875, and 6,660,910; Sathasivan et al.(1991) Plant Physiol 97:1044-1050; Ott et al. (1996) J Mol Biol263:359-368; and Fang et al. (1992) Plant Mol Biol 18:1185-1187). Inaddition, chemical resistance genes further include tryptophandecarboxylase which confers resistance to 4-methyl tryptophan (4-mT)(Goodijn et al. (1993) Plant Mol Biol 22:907-912); and bromoxynilnitrilase which confers resistance to bromoxynil. The selection markermay comprise cyanamide hydratase (Cah), see, for example, Greiner et al.(1991) Proc Natl Acad Sci USA 88:4260-4264; and Weeks et al. (2000) CropSci 40:1749-1754. Cyanamide hydratase enzyme converts cyanamide intourea, thereby conferring resistance to cyanamide. Any form or derivativeof cyanamide can be used as a selection agent including, but not limitedto, calcium cyanamide (Perlka® (SKW, Trotberg Germany) and hydrogencyanamide (Dormex® (SKW)). See also, U.S. Pat. Nos. 6,096,947, and6,268,547. Variants of cyanamide hydratase polynucleotides and/orpolypeptides will retain cyanamide hydratase activity. A biologicallyactive variant of cyanamide hydratase will retain the ability to convertcyanamide to urea. Methods to assay for such activity include assayingfor the resistance of plants expressing the cyanamide hydratase tocyanamide. Additional assays include the cyanamide hydratasecolorimetric assay (see, e.g., Weeks et al. (2000) Crop Sci40:1749-1754; and U.S. Pat. No. 6,268,547).

A polynucleotide indicates any nucleic acid molecule, and comprisesnaturally occurring, synthetic, and/or modified ribonucleotides,deoxyribonucleotides, and combinations of ribonucleotides anddeoxyribonucleotides. Polynucleotides encompass all forms of sequencesincluding, but not limited to, single-stranded, double-stranded, linear,circular, branched, hairpins, stem-loop structures, and the like. A DNAconstruct comprises a polynucleotide which, when present in the genomeof a plant, is heterologous or foreign to that chromosomal location inthe plant genome. In preparing the DNA construct, various fragments maybe manipulated to provide the sequences in a proper orientation and/orin the proper reading frame. Adapters or linkers may be employed to jointhe fragments. Other manipulations may be used to provide convenientrestriction sites, removal of superfluous DNA, or removal of restrictionsites. For example, in vitro mutagenesis, primer repair, restriction,annealing, resubstitutions, transitions, transversions, or recombinationsystems may be used. Polynucleotides, including polynucleotides ofinterest, markers, polynucleotides encoding a recombinase, recombinationsites, target sites, transfer cassettes, can be provided in a DNAconstruct. The construct can include 5′ and 3′ regulatory sequencesoperably linked to the appropriate sequences. The DNA construct caninclude in the 5′ to 3′ direction of transcription at least one of thefollowing, a transcriptional initiation region, a translationalinitiation region, the polynucleotide, and a transcriptional and/ortranslational termination region functional in plants. Alternatively,the DNA construct may lack at least one 5′ and/or 3′ regulatory element.For example, a DNA construct may be designed such that upon introductioninto a cell and in the presence of the appropriate recombinase arecombination event at the target site operably links the 5′ and/or 3′regulatory regions to the appropriate sequences of the DNA construct.Operably linked means that the nucleic acid sequences linked arecontiguous and comprise a functional linkage of the components.Regulatory elements can be used in a variety of ways depending on thepolynucleotide element, recombination site, transfer cassette and/ortarget site employed. In some examples intervening sequences can bepresent between operably linked elements and not disrupt the functionallinkage. For example, an operable linkage between a promoter and apolynucleotide of interest allows the promoter to initiate and mediatetranscription of the polynucleotide of interest. In some examples an ATGstart codon is operably linked to a recombination site. In someexamples, a recombination site is within an intron. The cassette mayadditionally contain at least one additional sequence to be introducedinto the plant. Alternatively, additional sequence(s) can be providedseparately. A DNA construct can be provided with a plurality ofrestriction sites and/or recombination sites for manipulation of thevarious components and elements. The DNA cassette may additionallycontain selectable marker genes.

A transcriptional initiation region may be native, analogous, foreign,or heterologous to the plant host or to the polynucleotide of interest,and may be a natural sequence, a modified sequence, or a syntheticsequence. A number of promoters can be used to express a codingsequence. A variety of promoters useful in plants is reviewed in Potenzaet al. (2004) In Vitro Cell Dev Biol Plant 40:1-22. In some examples,the promoter expressing the selection marker is active in the seed.Promoters active in the seed include constitutive promoters,developmental promoters, tissue specific promoters, inducible promoters,and the like. Constitutive promoters include for example, the corepromoter of the Rsyn7 promoter and other constitutive promotersdisclosed in WO99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35Spromoter (Odell et al. (1985) Nature 313:810-812); the MVV (mirabilismosaic virus) promoter (Dey & Maiti (1999) Plant Mol Biol 40:771-782);rice actin (McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin(Christensen et al. (1989) Plant Mol Biol 12:619-632, and Christensen etal. (1992) Plant Mol Biol 18:675-689); pEMU (Last et al. (1991) TheorAppl Genet 81:581-588); MAS (Velten et al. (1984) EMBO J 3:2723-2730);ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other constitutivepromoters include those disclosed in, e.g., U.S. Pat. Nos. 5,608,149;5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463;5,608,142; and 6,177,611. The promoter may be a tissue-preferredpromoter, e.g. to target enhanced expression within a particular planttissue. In some examples, a seed-preferred promoter is used to expressthe selection marker. Seed-preferred promoters include bothseed-specific promoters, active during seed development, as well asseed-germinating promoters, active during seed germination. See Thompsonet al. (1989) BioEssays 10:108. Seed-preferred promoters include, butare not limited to, cim1 (cytokinin-induced message); jip1(jasmonate-induced protein) (see WO02/42424); cZ19B1 (maize 19 kDazein); mi1ps, mi1ps3 (myo-inositol-1-phosphate synthase) (seeWO00/11177, WO02/42424, and U.S. Pat. No. 6,225,529); led (seeWO02/42424); oleosin (see Qu & Huang (1990) J Biol Chem 265:2238-2243,and Plant et al. (1994) Plant Mol Biol 25:193-202); rab17 (see Vilardellet al. 1990 Plant Mol Biol 14:423-432; Vilardell et al. 1991 Plant MolBiol 17:985-993; and Busk et al. 1997 Plant J 11:1285-1295); beanβ-phaseolin; napin; β-conglycinin; soybean lectin; cruciferin; maize 15kDa zein; 22 kDa zein; 27 kDa zein; waxy; shrunken1, shrunken2;globulin1, globulin2; end1, and end2 (WO00/12733); and the like. Inother examples, a chemical-regulated promoter is used. Achemical-regulated promoter can be used to modulate expression in theseed through the application of an exogenous chemical regulator. Thepromoter may be a chemical-inducible promoter, where application of thechemical induces gene expression, or a chemical-repressible promoter,where application of the chemical represses gene expression.Chemical-inducible promoters include, but are not limited to, the maizeIn2-2 promoter, activated by benzenesulfonamide herbicide safeners; themaize GST promoter, activated by hydrophobic electrophilic compounds(e.g., some pre-emergent herbicides); and the tobacco PR-1a promoter,activated by salicylic acid. Other chemical-regulated promoters ofinterest include steroid-responsive promoters (see, for example, theglucocorticoid-inducible promoter in Schena et al. (1991) Proc Natl AcadSci USA 88:10421-10425; and McNellis et al. (1998) Plant J 14:247-257)and tetracycline-inducible and tetracycline-repressible promoters (see,e.g., Gatz et al. (1991) Mol Gen Genet 227:229-237, and U.S. Pat. Nos.5,814,618, and 5,789,156).

The DNA construct can comprise expression units. An expression unitcomprises a promoter operably linked to another polynucleotide sequence.Expression units can have additional elements including, but not limitedto, introns, enhancers, leaders insulators, polynucleotides of interest,marker genes, recombination sites, translation initiation regions,termination regions, sequences encoding recombinases, etc. In addition,the DNA constructs can comprise transfer cassettes, target sites, or anyportions or combinations thereof. The DNA construct can be modified in avariety of ways, including site-specific recombination methods, toprovide a number of variations in the DNA construct. Polynucleotidesequences may be modified for expression in the plant. See, e.g.,Campbell & Gowri (1990) Plant Physiol 92:1-11. Methods for synthesizingplant-preferred genes include, e.g., U.S. Pat. Nos. 5,380,831,5,436,391, and Murray et al. (1989) Nucleic Acids Res 17:477-498.Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to average levels for a given host, ascalculated by reference to endogenous genes expressed in the host. Thesequence may also be modified to avoid secondary mRNA structures.Cassettes may additionally contain 5′ leader sequences in the DNAcassette which may act to enhance translation. Translation leadersinclude, e.g., picornavirus leaders such as EMCV leader (Elroy-Stein etal. (1989) Proc Natl Acad Sci USA 86:6126-6130); potyvirus leaders suchas TEV leader (Gallie et al. (1995) Gene 165:233-238), MDMV leader (Konget al. (1988) Arch Virol 143:1791-1799), and human immunoglobulinheavy-chain binding protein (BiP) (Macejak et al. (1991) Nature353:9094); untranslated leader from the coat protein mRNA of alfalfamosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625);tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in MolecularBiology of RNA, ed. Cech (Liss, New York), pp. 237-256); and maizechlorotic mottle virus leader (MCMV) (Lommel et al. (1991) Virology81:382-385). See also, Della-Cioppa et al. (1987) Plant Physiol84:965-968. Other methods or sequences known to enhance translation canalso be utilized, such as introns, and the like.

Polynucleotides of interest include, e.g., zinc fingers, kinases, heatshock proteins, transcription factors, DNA repair factors, agronomictraits, insect resistance, disease resistance, herbicide resistance,sterility, oil, protein, starch, digestibility, kernel size, maturity,nutrient composition, levels or metabolism, and the like. Insectresistance genes may encode resistance to pests such as rootworm,cutworm, European Corn Borer, and the like. Such genes include, e.g., B.thuringiensis toxic protein genes (U.S. Pat. Nos. 5,366,892; 5,747,450;5,736,514; 5,723,756; 5,593,881; Geiser et al. (1986) Gene 48:109); andthe like. Disease resistance traits include detoxification genes, suchas against fumonosin (U.S. Pat. No. 5,792,931); avirulence (avr) anddisease resistance (R) genes (Jones et al. (1994) Science 266:789;Martin et al. (1993) Science 262:1432; and Mindrinos et al. (1994) Cell78:1089); and the like. Herbicide resistance traits include genes codingfor resistance to herbicides including sulfonylurea-type herbicides(e.g., the S4 and/or Hra mutations in ALS), herbicides that act toinhibit action of glutamine synthase, such as phosphinothricin or basta(e.g., the bar gene), EPSPS (U.S. Pat. Nos. 6,867,293; 5,188,642; and5,627,061), GOX (Zhou et al. (1995) Plant Cell Rep 15:159-163), and GAT(U.S. Pat. No. 6,395,485). Antibiotic resistance genes may also be used,such as the nptII gene which encodes resistance to the antibioticskanamycin and geneticin. Sterility genes can also be used, for exampleas an alternative to detasseling, including male tissue-preferred genesand genes with male sterility phenotypes such as QM (e.g., U.S. Pat. No.5,583,210), kinases, and those encoding compounds toxic to either maleor female gametophytic development.

Reduction of the activity of specific genes, silencing and/orsuppression may be desired. Many techniques for gene silencing areknown, including but not limited to antisense technology (see, e.g.,Sheehy et al. (1988) Proc Natl Acad Sci USA 85:8805-8809; and U.S. Pat.Nos. 5,107,065; 5,453,566; and 5,759,829); cosuppression (e.g., Taylor(1997) Plant Cell 9:1245; Jorgensen (1990) Trends Biotech 8:340-344;Flavell (1994) Proc Natl Acad Sci USA 91:3490-3496; Finnegan et al.(1994) Bio/Technology 12:883-888; and Neuhuber et al. (1994) Mol GenGenet 244:230-241); RNA interference (Napoli et al. (1990) Plant Cell2:279-289; U.S. Pat. No. 5,034,323; Sharp (1999) Genes Dev 13:139-141;Zamore et al. (2000) Cell 101:25-33; Javier (2003) Nature 425:257-263;and, Montgomery et al. (1998) Proc Natl Acad Sci USA 95:15502-15507),virus-induced gene silencing (Burton et al. (2000) Plant Cell12:691-705; and Baulcombe (1999) Curr Op Plant Bio 2:109-113);target-RNA-specific ribozymes (Haseloff et al. (1988) Nature 334:585-591); hairpin structures (Smith et al. (2000) Nature 407:319-320;WO99/53050; WO02/00904; and WO98/53083); ribozymes (Steinecke et al.(1992) EMBO J 11:1525; U.S. Pat. No. 4,987,071; and, Perriman et al.(1993) Antisense Res Dev 3:253); oligonucleotide mediated targetedmodification (e.g., WO03/076574: and WO99/25853); Zn-finger targetedmolecules (e.g., WO01/52620; WO03/048345; and WO00/42219); and othermethods, or combinations of the above methods.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,or may be derived from another source. Convenient termination regionsare available from the Ti-plasmid of A. tumefaciens, such as theoctopine synthase and nopaline synthase termination regions. See alsoGuerineau et al. (1991) Mol Gen Genet 262:141-144; Proudfoot (1991) Cell64:671-674; Sanfacon et al. (1991) Genes Dev 5:141-149; Mogen et al.(1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158;Ballas et al. (1989) Nucleic Acids Res 17:7891-7903; and Joshi et al.(1987) Nucleic Acids Res 15:9627-9639.

Any method for introducing a sequence into a plant can be used, as longas the polynucleotide or polypeptide gains access to the interior of atleast one cell. Methods for introducing sequences into plants are knownand include, but are not limited to, stable transformation, transienttransformation, virus-mediated methods, and sexual breeding. Stablyincorporated indicates that the introduced polynucleotide is integratedinto a genome and is capable of being inherited by progeny. Transienttransformation indicates that an introduced sequence does not integrateinto a genome such that it is heritable by progeny from the host. Theplants and seeds employed may have a DNA construct stably incorporatedinto their genome. Any protocol may be used to introduce the DNAconstruct, any component of site-specific recombination systems, apolypeptide, or any other polynucleotide of interest. Providingcomprises any method that brings together any polypeptide and/or apolynucleotide with any other recited component(s). Any means can beused to bring together a target site, transfer cassette, and appropriaterecombinase, including, for example, stable transformation, transientdelivery, and sexual crossing (see, e.g., WO99/25884). In some examples,the recombinase may be provided in the form of the polypeptide or mRNA.A series of protocols may be used in order to bring together the variouscomponents. For instance, a cell can be provided with at least one ofthese components via a variety of methods including transient and stabletransformation methods; co-introducing a recombinase DNA, mRNA orprotein directly into the cell; employing an organism (e.g., a strain orline) that expresses the recombinase; or growing/culturing the cell ororganism carrying a target site, crossing to an organism expressing anactive recombinase protein, and selecting events in the progeny. Asimple integration pattern is produced when the transfer cassetteintegrates predominantly at the target site, and at less than about 25,20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 random position(s) in thegenome. Any promoter, including constitutive, inducible,developmentally, temporal, and/or spatially regulated promoter, etc.,that is capable of regulating expression in the organism may be used.

Transformation protocols as well as protocols for introducingpolypeptides or polynucleotide sequences into plants may vary dependingon the type of plant or plant cell targeted for transformation. Suitablemethods of introducing polypeptides and polynucleotides into plant cellsinclude microinjection (Crossway et al. (1986) Biotechniques 4:320-334,and U.S. Pat. No. 6,300,543), electroporation (Riggs et al. (1986) ProcNatl Acad Sci USA 83:5602-5606, Agrobacterium-mediated transformation(U.S. Pat. Nos. 5,563,055; and 5,981,840), direct gene transfer(Paszkowski et al. (1984) EMBO J 3:2717-2722), and ballistic particleacceleration (U.S. Pat. Nos. 4,945,050; 5,879,918; 5,886,244; and5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture:Fundamental Methods, ed. Gamborg & Phillips (Springer-Verlag, Berlin);McCabe et al. (1988) Biotechnology 6:923-926); and Led transformation(WO00/28058). Also see Weissinger et al. (1988) Ann Rev Genet22:421-477; Sanford et al. (1987) Particulate Science and Technology5:27-37 (onion); Christou et al. (1988) Plant Physiol 87:671-674(soybean); Finer & McMullen (1991) In Vitro Cell Dev Biol 27P:175-182(soybean); Singh et al. (1998) Theor Appl Genet 96:319-324 (soybean);Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988)Proc Natl Acad Sci USA 85:4305-4309 (maize); Klein et al. (1988)Biotechnology 6:559-563 (maize); U.S. Pat. Nos. 5,240,855; 5,322,783;and, 5,324,646; Klein et al. (1988) Plant Physiol 91:440-444 (maize);Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-VanSlogteren et al. (1984) Nature 311:763-764; U.S. Pat. No. 5,736,369(cereals); Bytebier et al. (1987) Proc Natl Acad Sci USA 84:5345-5349(Liliaceae); De Wet et al. (1985) in The Experimental Manipulation ofOvule Tissues, ed. Chapman et al. (Longman, New York), pp. 197-209(pollen); Kaeppler et al. (1990) Plant Cell Rep 9:415-418; and Kaeppleret al. (1992) Theor Appl Genet 84:560-566 (whisker-mediatedtransformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505(electroporation); Li et al. (1993) Plant Cell Rep 12:250-255; Christou& Ford (1995) Ann Bot 75:407-413 (rice); and Osjoda et al. (1996) NatBiotechnol 14:745-750 (maize via A. tumefaciens).

The polynucleotide may be introduced into plants by contacting plantswith a virus, or viral nucleic acids. Generally, such methods involveincorporating a desired polynucleotide within a viral DNA or RNAmolecule. The sequence may initially be synthesized in a viralpolyprotein and later processed in vivo or in vitro to produce a desiredprotein. Useful promoters encompass promoters utilized for transcriptionby viral RNA polymerases. Methods for introducing polynucleotides intoplants and expressing a protein encoded, involving viral DNA or RNAmolecules, are known, see, e.g., U.S. Pat. Nos. 5,889,191; 5,889,190;5,866,785; 5,589,367; 5,316,931; and Porta et al. (1996) Mol Biotech5:209-221.

Various components, including those from a site-specific recombinationsystem, can be provided to a plant using a variety of transient methods.Such transient transformation methods include, but are not limited to,providing recombinase polypeptide directly, providing a recombinasemRNA, using a non-integrative method, or providing low levels of DNAinto the plant. Such methods include, for example, microinjection,particle bombardment, viral vector systems, and/or precipitation of thepolynucleotide wherein transcription occurs from the particle-bound DNAwithout substantive release or integration into the genome, such methodsgenerally use particles coated with polyethylimine, (see, e.g., Crosswayet al. (1986) Mol Gen Genet 202:179-185; Nomura et al. (1986) Plant Sci44:53-58; Hepler et al. (1994) Proc Natl Acad Sci USA 91:2176-2180; andHush et al. (1994) J Cell Sci 107:775-784).

The term plant includes plant cells, plant protoplasts, plant celltissue cultures from which a plant can be regenerated, plant calli,plant clumps, and plant cells that are intact in plants or parts ofplants such as embryos, pollen, ovules, seeds, leaves, flowers,branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips,anthers, and the like. Progeny, variants, and mutants of the regeneratedplants are also included.

Any plant species can be used with the methods and compositions,including, but not limited to, monocots and dicots. Examples of plantgenuses and species include, but are not limited to, corn (Zea mays),Brassica sp. (e.g., B. napus, B. rapa, B. juncea), castor, palm, alfalfa(Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum(Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet(Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet(Setaria italica), finger millet (Eleusine coracana)), sunflower(Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticumaestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato(Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypiumbarbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava(Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera),pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobromacacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Perseaamericana), fig (Ficus casica), guava (Psidium guajava), mango(Mangifera indica), olive (Olea europaea), papaya (Carica papaya),cashew (Anacardium occidentale), macadamia (Macadamia integrifolia),almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane(Saccharum spp.), Arabidopsis thaliana, oats (Avena spp.), barley(Hordeum spp.), leguminous plants such as guar beans, locust bean,fenugreek, garden beans, cowpea, mungbean, fava bean, lentils, andchickpea, vegetables, ornamentals, grasses and conifers. Vegetablesinclude tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactucasativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Pisium spp., Lathyrus spp.), and Cucumis species suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum. Conifers include pines, forexample, loblolly pine (Pinus taeda), slash pine (Pinus elliotii),ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), andMonterey pine (Pinus radiata), Douglas fir (Pseudotsuga menziesii);Western hemlock (Tsuga canadensis), Sitka spruce (Picea glauca), redwood(Sequoia sempervirens), true firs such as silver fir (Abies amabilis)and balsam fir (Abies balsamea), and cedars such as Western red cedar(Thuja plicata) and Alaska yellow cedar (Chamaecyparis nootkatensis).

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Rep 5:81-84. These plants may then be grown andself-pollinated, backcrossed, and/or outcrossed, and the resultingprogeny having the desired characteristic identified. Two or moregenerations may be grown to ensure that the characteristic is stablymaintained and inherited and then seeds harvested. In this mannertransformed/transgenic seed having the recited DNA construct stablyincorporated into their genome are provided. A plant and/or a seedhaving stably incorporated the DNA construct can be furthercharacterized for expression, site-specific integration potential,agronomics, and copy number (see, e.g., U.S. Pat. No. 6,187,994).

Sequence identity may be used to compare the primary structure of twopolynucleotides or polypeptide sequences. Sequence identity measures theresidues in the two sequences that are the same when aligned for maximumcorrespondence. Sequence relationships can be analyzed usingcomputer-implemented algorithms. The sequence relationship between twoor more polynucleotides, or two or more polypeptides can be determinedby computing the best alignment of the sequences, and scoring thematches and the gaps in the alignment, which yields the percent sequenceidentity, and the percent sequence similarity. Polynucleotiderelationships can also be described based on a comparison of thepolypeptides each encodes. Many programs and algorithms for comparisonand analysis of sequences are known. Unless otherwise stated, sequenceidentity/similarity values provided herein refer to the value obtainedusing GAP Version 10 (GCG, Accelrys, San Diego, Calif.) using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix (Henikoff & Henikoff (1992) Proc. Natl. Acad.Sci. USA 89:10915-10919). GAP uses the algorithm of Needleman & Wunsch(1970) J Mol Biol 48:443-453, to find the alignment of two completesequences that maximizes the number of matches and minimizes the numberof gaps.

Variant polynucleotides include polynucleotides having at least onedeletion, addition, and/or substitution in at least one of the 5′ end,3′ end, and/or internal sites including introns or exons, as compared tothe native polynucleotide. Variant polynucleotides include naturallyoccurring variants as well as synthetically derived polynucleotides, forexample, those generated using site-directed mutagenesis. Conservativevariants include sequences that maintain their function, encode the samepolypeptide, or encode a variant polypeptide with substantially similaridentity, function, and/or activity as the native polynucleotide.Variants can be identified with known techniques, for example,polymerase chain reaction (PCR), and/or hybridization techniques.Generally, variants of a particular polynucleotide will have at leastabout 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to thatparticular polynucleotide. Variant polynucleotides can also be evaluatedby comparison of the percent sequence identity between the polypeptidesencoded using standard alignment programs and parameters. When evaluatedby comparison of the percent sequence identity shared by thepolypeptides each encodes, the percent sequence identity between the twoencoded polypeptides is typically at least about 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more sequence identity.

Variant proteins include proteins having at least one deletion,addition, and/or substitution in at least one of the N-terminal end,C-terminal end, and/or an internal site, as compared to the nativepolypeptide. Variant proteins possess the desired biological activity ofthe protein. Variants include naturally occurring polypeptides, as wellas those generated by human manipulation. Biologically active variantsof a protein typically have at least about 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity to the amino acid sequence for the native proteinas determined by sequence alignment programs. A biologically activevariant of a protein may differ from that protein by as few as 1-15amino acid residues. Conservative substitutions generally refer toexchanging one amino acid with another having similar properties. Forexample, the model of Dayhoff et al. (1978) Atlas of Protein Sequenceand Structure (Natl Biomed Res Found, Washington, D.C.) providesguidance on amino acid substitutions that are not expected to affect thebiological activity of the protein.

Variant polynucleotides and proteins encompass sequences derived frommutagenic and/or recombinogenic procedures, such as mutagenesis and/orDNA shuffling. Methods for mutagenesis, nucleotide sequence alterations,and DNA shuffling are known (see, e.g., Kunkel (1985) Proc Natl Acad SciUSA 82:488-492; Kunkel et al. (1987) Methods Enzymol 154:367-382; U.S.Pat. No. 4,873,192; Walker & Gaastra, eds. (1983) Techniques inMolecular Biology (MacMillan Publ. Co., NY); Stemmer (1994) Proc NatlAcad Sci USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameriet al. (1997) Nat Biotechnol 15:436-438; Moore et al. (1997) J Mol Biol272:336-347; Zhang et al. (1997) Proc Natl Acad Sci USA 94:4504-4509;Crameri et al. (1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793,and 5,837,458). For example, one or more different recombinase codingsequences can be manipulated to create and select a new recombinaseprotein possessing the desired properties. DNA shuffling typicallygenerates libraries of recombinant polynucleotides from a population ofrelated sequences, which are homologously recombined in vitro or invivo. Generally, any modification(s) to a polynucleotide encoding apolypeptide should not alter the reading frame, or create and/or alterDNA or mRNA secondary structure. See, EP Patent Application PublicationNo. 75,444.

Fragments and variants of recombination sites, recombinases, selectionmarkers, and nucleotide sequences of interest can be used, and unlessotherwise stated, indicate that the variant or fragment retains at leastsome of the activity/function of the original composition. For example,for a polynucleotide encoding a protein, a fragment of a polynucleotideencodes a polypeptide protein that retains at least some of thebiological activity of the full-length protein. Fragments of apolynucleotide may range from at least about 20 nucleotides, about 50nucleotides, about 100 nucleotides, up to the full-lengthpolynucleotide. A fragment of a polynucleotide that encodes abiologically active portion of a protein typically encodes at least 15,25, 30, 50, 100, 150, 200, 250, 300, 325, 350, 375, 400, 420, or 450contiguous amino acids, or any integer in this range up to and includingthe total number of amino acids present in a full-length protein. Abiologically active fragment of a polypeptide can be prepared byisolating a portion of one of the polynucleotides encoding the portionof the polypeptide of interest, expressing the protein fragment, andassessing the activity. Alternatively, a biologically active fragment ofa polypeptide can be produced by selective chemical or proteolyticcleavage of the full-length polypeptide, and the activity measured. Forexample, polynucleotides that encode fragments of a recombinasepolypeptide may comprise at least 16, 20, 50, 75, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,100,1,200, 1,300, or 1,400 nucleotides, or any integer in this range up toand including the total number of nucleotides of a full-lengthpolynucleotide. In addition, fragments of a recombination site retainthe biological activity of the recombination site, undergoing arecombination event in the presence of the appropriate recombinase.Fragments of a recombination site may range from at least about 5, 10,15, 20, 25, 30, 35, 40 nucleotides, up to the full-length of arecombination site. For example, a full-length FRT, lox, attB, and attPsites are known and range from about 50 nucleotides to about 250nucleotides, and fully active minimal sites are known and range fromabout 20, 25, 30, 35, 40, 45, and 50 nucleotides. Biologically activevariants of a recombination site retain the biological activity of therecombination site, undergoing a recombination event in the presence ofthe appropriate recombinase. Biologically active variants ofrecombination sites have at least one substitution, addition, ordeletion as compared to a full-length recombination site.

Assays to measure the biological activity of recombination sites andrecombinases are known (see, e.g., Senecoll et al. (1988) J Mol Biol201:406-421; Voziyanov et al. (2002) Nucleic Acids Res 30:7; U.S. Pat.No. 6,187,994; WO01/00158; Albert et al. (1995) Plant J 7:649-659;Hartang et al. (1998) J Biol Chem 273:22884-22891; Saxena et al. (1997)Biochim Biophy Acta 1340:187-204; and Hartley et al. (1980) Nature280-860-864). Assays for recombinase activity measure the overallactivity of the enzyme on DNA substrates containing recombination sites.For example, to assay for FLP activity, inversion of a DNA sequence in acircular plasmid containing two inverted FRT sites can be detected as achange in position of restriction enzyme sites (see, e.g., Vetter et al.(1983) Proc Natl Acad Sci USA 80:7284). Alternatively, excision of DNAfrom a linear molecule or intermolecular recombination frequency inducedby the enzyme may be assayed (see, e.g., Babineau et al. (1985) J BiolChem 260:12313; Meyer-Leon et al. (1987) Nucleic Acids Res 15:6469; andGronostajski et al. (1985) J Biol Chem 260:12328). Recombinase activitymay also be measured by excision of a sequence flanked by recombinogenicFRT sites to activate an assayable marker gene. Similar assays are usedto measure the activity of recombination sites.

The following examples are offered by way of illustration and not by wayof limitation.

Example 1 Method of Seed Priming with Dormex®

For initial determination of the parameters, ranges, and/or conditionsfor seed priming, non-transgenic seed, transgenic seed transformed witha construct comprising Ubi::moCAH produced throughAgrobacterium-mediated transformation were used. In further tests,transgenic seed were used comprising a putative targeted insertion andactivation of the selectable marker were generated essentially asdescribed in Example 3.

A. Determining Soaking Time

Non-transgenic seeds from two inbred corn lines, N46 and P38 were usedto evaluate the conditions for seed priming with the selective agent.For each treatment, 24 seeds each from N46 and P38 were soaked in 10 mlof water, or 0.5%, 1%, or 5% Dormex® in water for 10 min., 30 min., 1hr, and 2 hrs, with vacuum applied. Following the soaking period, theDormex® solution was removed and the seeds were placed into apollination bag. A fan was placed in front of the seed bags, and theseeds were dried for times ranging from overnight to 2 days. The driedseeds were planted into flats to germinate. Table 2 provides germinationresults for a test with inbred N46.

TABLE 2 Corn % Soaking Total # % Line Dormex ® Time Seed SeedlingsGermination N46 0 10 min. 24 24 100 N46 0.5 10 min. 24 24 100 N46 1 10min. 24 23 100 N46 5 10 min. 24 19 79 N46 0 30 min. 24 24 100 N46 0.5 30min. 24 23 96 N46 1 30 min. 24 21 88 N46 5 30 min. 24 9 38 N46 0 1 hr.24 24 100 N46 0.5 1 hr. 24 22 92 N46 1 1 hr. 24 14 58 N46 5 1 hr. 24 2 8N46 0 2 hr. 24 23 96 N46 0.5 2 hr. 24 19 79 N46 1 2 hr. 24 13 54 N46 5 2hr. 24 1 4

B. Determining Selective Agent Concentration

1. Non-Transgenic Seed

Experiments were conducted testing ranges of Dormex® concentrationsranging from 0.1%-30% v/v. Seed from inbred maize lines N46 and P38 wereused, typically with a priming time of at least 2 hours, and overnightdrying post-priming. Germination results of tests of Dormex®concentration are shown in Table 3.

TABLE 3 Corn Line % Dormex ® Total Seed % Germination P38 0 10 100 P380.1 10 40 P38 0.3 10 0 P38 0.5 10 0 P38 0.7 10 0 P38 1 10 0 N46 0 10 100N46 0.1 10 20 N46 0.3 10 0 N46 0.5 10 0 N46 0.7 10 0 N46 1 10 0 N46 0 9696 N46 5 96 0 N46 10 96 0 N46 15 92 0 N46 30 96 0

2. Transgenic Seed

A range of Dormex® concentrations and treatment times were tested,ranging from 0.5%-5% v/v. Seed from each independent transgenic event,hemizygous for ubi::moCah, were prepared by placing seed into mesh bags.The seeds were then placed into a Dormex® solution+0.4% glycerol (1 Lcomprised: desired volume of Dormex®, 4 ml glycerol and steriledeionized water to 1 L) to just cover seed. The seeds were incubated for18 hours in the dark at 20° C. Following the incubation, the seeds wereremoved from the Dormex® solution, rinsed with water, allowed to dry for1-3 days before germinating. Typically, optimal germination conditionswere obtained by wrapping the dry seed in sterile moistened germinationpaper and incubation in the dark at 37° C. for 3-4 days. Germinatedseedlings were further screened by PCR to confirm the presence of themoCAH gene. In the first trial, events with higher copy number tended toshow lower Dormex® resistance, a possible indication of transgenerearrangement. Events with 2 or fewer copies of the transgene werere-tested. TABLE 4 presents the percentage of germinated seedlings usingvarying Dormex® concentrations for priming for the two combined tests.Some escapes could be found at Dormex® lower concentrations lower than1%, as determined by PCR verification for the moCah gene.

TABLE 4 Dormex ® concentration Event 0% 0.3% 0.5% 0.7% 1% 1.5%N46_2237612 100 10 0 0 0 0 P38_2242574 100 80 0 0 0 0 P38_2145089 87 10077 67 63 50 N46_2145082 70 30 37 43 23 27 P38_2171582 80 60 54 64 36 40N46_2171578 87 93 63 93 60 73 N46_2145072 70 60 72 80 80 32 P38_223762590 10 0 0 0 0 N46_1887662 80 80 80 80 80 50

In a separate experiment, transgenic inbred lines N46 and P38,comprising Ubi::moCah, and non-transgenic controls were primed in 0-5%v/v Dormex® solution, with no additives, for 3 hours, transferred tomesh bags, dried overnight in front of a fan, and planted in flats forgermination. The presence of moCah was verified by enzyme assay &/or PCRanalysis of selected germinated seedlings. The results are shown inTable 5.

TABLE 5 Corn % Total % PCR Line Dormex ® Seed Germination Enzyme moCah+N46 0 48 98 0/8 0 N46 1 48 6 0/2 0 N46 3 44 0 — — N46 5 47 0 — —N46_1422777 0 48 67 — — N46_1422777 1 46 27  0/13 — N46_1422777 5 43 70/3 3/3 N46_1422780 5 36 44  1/16 10/10 N46_2025731 0 48 98 — —N46_2025731 3 48 54 21/26 — N46_2025731 5 48 19 7/9 7/7 P38_1370184 0 48100 — — P38_1370184 1 48 77  4/37 — P38_1370184 3 48 67  3/33 —P38_1370184 5 48 31  5/15   9/10** P38_1445805 5 35 14 5/5 — P38_21715825 7 29 2/2 2/2 P38 0 41 93 0/7 0/2 P38 1 48 0 — — P38 3 48 0 — — P38 532 0 — — **DNA isolation failed from one sample

C. Seed Priming Selection of Recombinant Target

Non-transgenic and transgenic maize seeds were placed in nylon net bagsand soaked in 3% or 5% Dormex® solution for 3 hrs. Two lots of Dormex®were used in this experiment. The seeds were removed from the bags,placed on a flat surface and dried for 3 days, some dried primed seedwere then planted into flats to germinate, and remaining primed seedwere stored. Selected germinated seedlings were further analyzed forcyanamide hydratase enzyme activity, and by PCR to confirm the linkageof the ubiquitin promoter to the moCAH polynucleotide. These results areprovided in Table 6. Samples N46-1 through P38-12 are non-transgeniccontrols, no germination was observed after priming with Dormex®.Samples N46-13 through N46-21 are transgenic seed, N46-21 is atransgenic control primed with water, N46-13 and N46-14 are from atransgenic target line comprising PHP17797, which lacks the moCAHselectable marker, the remaining samples are paired samples of targetedseed testing linkage and activation of donor moCAH selectable marker tothe target ubiquitin promoter.

TABLE 6 Corn Total % Line Dormex ® Lot seed Planted Seedling GerminationEnzyme PCR+ N46-1 3% 1 24 12 0 0 — — N46-2 3% 1 100 12 0 0 — — N46-3 3%2 24 12 0 0 — — N46-4 3% 2 100 12 0 0 — — N46-5 5% 2 24 12 0 0 — — N46-65% 2 100 12 0 0 — — P38-7 3% 1 24 12 0 0 — — P38-8 3% 1 100 12 0 0 — —P38-9 3% 2 24 12 0 0 — — P38-10 3% 2 100 12 0 0 — — P38-11 5% 2 24 12 00 — — P38-12 5% 2 100 12 0 0 — — N46-13 3% 2 18 9 0 0 — — N46-14 5% 2 189 0 0 — — P38-15 3% 2 50 24 13 54 12/13 12/13 P38-16 5% 2 43 24 6 25 6/66/6 N46-17 3% 2 25 12 5 42 5/5 5/5 N46-18 5% 2 25 12 1 8 1/1 1/1 N46-193% 2 25 12 1 8 0/1 0/1 N46-20 5% 2 25 12 0 0 — — N46-21 0% 2 12 12 11 92 0/11  0/11

The data demonstrate that escapes were possible at priming times lessthan 3 hours. In addition, variable Dormex® tolerance was detected inthe different corn genotypes tested. Specifically, it was found that N46wild type seeds are more tolerant to Dormex® than P38 wild type seeds.It was also observed that the concentration of Dormex® effective toselect events by seed priming was about 1/10 of the concentrationtypically used for leaf painting or spraying plants (600 mM). Theconcentration of selective agent and treatment time should be evaluatedfor seeds of different genotypes.

D. Drying the Primed Seed

After priming, the seeds may be planted and allowed to germinate, or theprimed seeds can be dried for storage. The appropriate conditions(temperature, relative humidity, and time) for the drying process willvary depending on the seed and can be determined empirically. See, forexample, Jeller et al. (2003) Braz J Biol 63:61-68. Drying the primedseed includes a superficial drying of the seed or, alternatively, dryingthe seed back to its original water content. Alternatively, primed seedcan be germinated immediately after priming without drying. Further, thedried seeds can be immediately germinated or can be stored underappropriate conditions. All of the above methods were tested and havebeen used. The method adopted was to dry the seed in a drying oven, orin the greenhouse, for 3 days followed by storage at a cool drytemperature for up to a month.

Similar testing can be done to determine appropriate priming conditionsfor this and other selective agents in other corn inbred lines, cornhybrids, and other seeds such as soybean, rice, cotton, canola, etc.

Seed from non-transgenic inbred lines N46 and P38 primed with Dormex®concentrations ranging from 0-1% v/v, in parallel with one test shown inTable 3, and germinated after 6 days of drying & storage. Thegermination results are shown in Table 7.

TABLE 7 Corn Line % Dormex ® Total Seed % Germination P38 0 10 100 P380.1 10 60 P38 0.3 10 0 P38 0.5 10 0 P38 0.7 10 0 P38 1 10 0 N46 0 10 80N46 0.1 10 100 N46 0.3 10 0 N46 0.5 10 0 N46 0.7 10 0 N46 1 10 0

Seed from the experiment shown in Table 6 were stored for one month, andthen planted in flats to germinate. The germination results are shown inTable 8. Samples N46-1 through P38-12 are non-transgenic controls, nogermination was observed after priming with Dormex®. Samples N46-13through N46-21 are transgenic seed, N46-21 is a transgenic controlprimed with water, N46-13 and N46-14 are from a transgenic target linecomprising PHP17797, which lacks the moCAH selectable marker, theremaining samples represent paired samples of seed from targeted linkageand activation of moCAH selectable marker from the donor, to theubiquitin promoter in the target.

TABLE 8 Corn Line % Dormex Lot Total Seed % Germination N46-1 3 1 12 0N46-2 3 1 12 0 N46-3 3 2 12 0 N46-4 3 2 12 0 N46-5 5 2 12 0 N46-6 5 2 120 P38-7 3 1 12 0 P38-8 3 1 12 0 P38-9 3 2 12 0 P38-10 3 2 12 0 P38-11 52 12 0 P38-12 5 2 12 0 N46-13 3 2 9 0 N46-14 5 2 9 0 P38-15 3 2 26 35P38-16 5 2 19 32 N46-17 3 2 13 23 N46-18 5 2 13 0 N46-19 3 2 13 0 N46-205 2 13 0 N46-21 0 — 12 83

E. Testing Priming Matrix Additives

A variety of additives can be included in the priming matrix, includingsurfactants, additional selective agents, fungicides, agents to modifyosmotic potential, osmotic protectants, agents to extend storage shelflife, agents to enhance coating and/or perfusion, and the like.

1. Dormex®+DMSO

Non-transgenic maize seed from inbred N46 were primed with 0, 5, 10, 15,or 30% Dormex® solutions with and without 1% DMSO. For each treatment,96 seeds were soaked in 50 ml of Dormex® solution in a petri dish for 3hours at room temperature. After priming the seeds were transferred intopollination bags, and dried overnight in front of a fan. Dried primedseed were then planted into flats to germinate. Results are shown inTable 9.

TABLE 9 Treatment Total Seed Seedlings Germination % Water 96 92 96 5%Dormex ® 96 0 0 10% Dormex ® 96 0 0 15% Dormex ® 92 0 0 30% Dormex ® 960 0 1% DMSO 95 88 93 5% Dormex ®, 1% DMSO 96 0 0 10% Dormex ®, 1% DMSO96 0 0 15% Dormex ®, 1% DMSO 96 0 0 30% Dormex ®, 1% DMSO 96 0 0

2. Dormex®+Tween-20

Fifty F1 seeds from crosses of inbred donor lines, comprising PHP18000,to target lines, comprising PHP17797, were primed in 2.2% Dormex®+0.4%Tween-20 for 3 hours, then dried overnight before planting 25 seed insoil, with the remaining 25 primed seed germinated at 32° C. usinggermination paper. Linkage of the moCah gene from the donor to the ubipromoter in the target was confirmed by PCR, all germinated seeds werePCR positive for ubi::moCah linkage. Results are shown in Table 10.

TABLE 10 % Germination Corn Line Soil Paper N46_3313853 72 84N46_3313686 44 80 P38_3312969 4   8** P38_3312982 0  1 **Germinationvery slow, at a later time point in the paper germination test 20% ofseeds had germinated.

In another experiment, F1 seeds from crosses of N46 inbred donor lines,comprising PHP18000, to target lines, comprising PHP17797, were primedin 2.2% Dormex®+0.4% Tween-20 for 3 hours, then dried overnight beforeplanting in soil. The germination results are shown in Table 11.

TABLE 11 Corn Line Total Seed % Germination N46_3313645 2 0 N46_33136754 25 N46_3313809 4 0 N46_3313891 4 75 N46_3313903 5 0 N46_3313866 6 17N46_3313601 7 0 N46_3313823 7 29 N46_3313761 10 0 N46_3313801 10 30N46_3313572 12 0 N46_3313712 13 0 N46_3313723 23 48 N46_3313901 13 23N46_3313821 14 0 TOTAL 134 17

Priming with 2-10% v/v Dormex®+0.4% Tween-20, and paper germination at25° C. or 32° C. was tested using non-transgenic N46 and P38 seed.Germination was checked at 3, 4, and/or 5 days after planting. Percentgermination results are shown in Table 12.

TABLE 12 Paper Germination Corn % Dormex Total 25° C. 32° C. Line 0.4%Tw-20 Seed 4 day 5 day 3 day 5 day N46 2   20 0 0 0 0 N46 2.2 20 0 0 0 0N46 2.5 20 0 0 0 0 N46 3   20 0 0 0 0 N46 5   20 0 0 0 0 N46 10   20 0 00 0 N46  0** 20 80 80 85 90 P38 2   20 0 0 0 0 P38 2.2 20 0 0 0 0 P382.5 20 0 0 0 0 P38 3   20 0 0 0 0 P38  0** 20 95 95 100 100 **Water onlytreatment, no Tween-20 used.

Example 2 Seed Priming with Glyphosate

Maize seeds are prepared by placing seed into a mesh bag. The seeds arethen placed into a glyphosate solution to just cover seed. The seeds areincubated for varying periods of time in the dark at 20° C. Followingthe incubation, the seeds are removed from the glyphosate solution,rinsed with water, and allowed to dry for 1-3 days and then stored, orplaced in flats to germinate.

A range of glyphosate concentrations, treatment times are tested on atleast two corn inbreds. For example, glyphosate concentrations rangingfrom about 0.3 mM to 80 mM are used, which encompasses the concentrationrange used for spraying plants, with 18 mM (0.5%) being approximately 1×application rate. A range of soak times from about 10 minutes to about 5hours or more is also tested, as are the conditions for drying andstorage of primed seed.

Similar testing, as done in Examples 1-2, can be done to determineappropriate priming conditions for this and other selective agents inother corn inbred lines, corn hybrids, and other seeds such as soybean,rice, cotton, canola, etc.

Example 3 Generating and Selecting a Targeted Seed

Any method can be used to provide a target site, a transfer cassette,and a recombinase to a plant to generate targeted seeds from any plantcomprising a polynucleotide encoding a selectable marker operably linkedto a promoter, which can be identified and selected by priming the wholepopulation of seeds with the selective agent. Constructs are generatedbased on the method of transformation to be used. Further, any selectiveagent can be used, under a variety of conditions.

Events can further be characterized for copy number, homozygosity, geneexpression, integrity of the inserted construct, and the like. Plantscan be evaluated for any phenotypic effect due to the insertion of thetarget site, transfer cassette, recombinase, or other polynucleotides ofinterest. Further, plants can be crossed or outcrossed to generatedhomozygous lines, or transfer the component to another plant line.

A. Transformation Vectors

Constructs comprising the transfer cassette or the target site aregenerated and used to transform plants to establish donor and acceptorplant lines, respectively. The target site and the transfer cassette aredesigned to allow for the selection of a targeted integration event bypriming or soaking seed from putative targeted events with a selectiveagent. In this example, the target site and transfer cassette aredesigned to replace a FLP recombinase encoding sequence with a cyanamidehydratase encoding sequence, thereby operably linking and activating theselectable marker, and deactivating the recombinase.

Optionally, standard techniques can be used to provide the recombinase,for example, providing a separate construct encoding the FLP recombinaseoperably linked to a promoter, placing the FLP coding region in adifferent location in the target construct, or placing the FLP codingregion on the transfer cassette, or providing FLP DNA, mRNA, or proteintransiently.

i. Agrobacterium Vectors

Agrobacterium binary plasmids were made using the hybrid systemdescribed by Komari et al. ((1996) Plant J 10:165-174). Derivatives ofpSB11 were built as intermediate T-DNA constructs containing the desiredconfiguration between the T-DNA border sequences. Plasmid pSB11 wasobtained from Japan Tobacco Inc. (Tokyo, Japan). Construction of pSB11from pSB21, and construction of pSB21 from starting vectors, isdescribed by Komari et al. ((1996) Plant J 10:165-174). Description ofintegration of the T-DNA plasmid into the superbinary plasmid pSB1 byhomologous recombination can be found in EP672752 A1. The plasmid pSB1was also obtained from Japan Tobacco Inc. These plasmids were used forAgrobacterium-mediated transformation after making the co-integrant inLBA4404. Electro-competent cells of the Agrobacterium strain LBA4404harboring pSB1 were created using the protocol as described by Lin(1995) in Methods in Molecular Biology, ed. Nickoloff, J. A. (HumanaPress, Totowa, N.J.). Cells and DNA were prepared for electroporation bymixing 1 μl plasmid DNA (˜100 ng) with 20 μl of competent cells in aLife Technologies (now Whatman Biometra) 0.15 cm electrode gap cuvette(Whatman Biometra #11608-031). Electroporation was performed in aCell-Porator Electroporation device using the Pulse Control unit(Whatman Biometra #11604-014) at the 330 μF setting along with theVoltage Booster (Whatman Biometra #11612-017) set at 4 kW. The systemdelivers approximately 1.8 kV to the Agrobacterium cells. Successfulrecombination was verified by restriction analysis of the co-integrantplasmid following isolation and transformation back into E. coli DH5αcells for amplification.

PHP17797 target and FLPm expression vector comprises: RB-ubi pro::ubiintron/FRT1/ubi intron:: FLPm::pinII-CaMV35S enh::CaMV35S pro::adh1intron::bar::pinII::FRT5-LB

The maize ubi intron is approximately 1010 nucleotides long. Forconstruction of target site vector PHP17797, the 48-nucleotide FRT1 sitewas introduced, along with flanking restriction sites HindIII (5′) andBgIII (3′), between nucleotides 974 and 975 for a final approximatelength of 1070 nucleotides. FRT site introduction can be done variety ofways, such as those described in U.S. Pat. No. 6,187,994. In PHP17797,the FRT5 site is in a short (65 nt) sequence between pinII and LB.Spectinomycin resistance is outside the T-DNA borders in the vectorbackbone. The final desired molecule was isolated from spectinomycinresistant E. coli isolates and verified by restriction digestionanalysis.

PHP18000 moCAH transfer cassette vector comprises: RB-FRT1/ubiintron::moCAH::pinII-35CaMV enh::ubi pro::ubi 5′ UTR::ubi intron::GFPmexon1::ST-LS1 intron::GFPm exon2::pinII::FRT5-LB

PHP18000 comprises FRT1 linked to a truncated ubi intron such that ithas the same sequence and configuration as the target (PHP17797),recombination with the target regenerates the ubi intron. The GFPmcoding sequence contains the ST-LS1 intron from potato. Spectinomycinresistance is outside the T-DNA borders in the vector backbone. Thefinal desired molecule was isolated from spectinomycin resistant E. coliisolates and verified by restriction digestion analysis.

B. Generation of Donor and Acceptor Plants

Transgenic donor and acceptor plant lines can be established via anytransformation method, for example Agrobacterium mediated infection orparticle bombardment.

i. Agrobacterium Mediated Transformation

Agrobacterium mediated transformation of maize is performed essentiallyas described by Zhao (WO98/32326). Briefly, immature embryos areisolated from maize and the embryos contacted with a suspension ofAgrobacterium containing a T-DNA, where the bacteria are capable oftransferring the nucleotide sequence of interest to at least one cell ofat least one of the immature embryos.

Step 1: Infection Step. In this step the immature embryos are immersedin an Agrobacterium suspension for the initiation of inoculation.Step 2: Co-cultivation Step. The embryos are co-cultured for a time withthe Agrobacterium.Step 3: Resting Step. Optionally, following co-cultivation, a restingstep may be performed. The immature embryos are cultured on solid mediumwith antibiotic, but without a selecting agent, for elimination ofAgrobacterium and for a resting phase for the infected cells.Step 4: Selection Step. Inoculated embryos are cultured on mediumcontaining a selective agent and growing transformed callus isrecovered. The immature embryos are cultured on solid medium with aselective agent resulting in the selective growth of transformed cells.Step 5: Regeneration Step. Calli grown on selective medium are culturedon solid medium to regenerate the plants.

ii. Particle Bombardment of Maize

Immature maize embryos are bombarded with a DNA construct comprising thetransfer cassette or comprising the target site. The recombinase can beprovided on the target site construct, transfer cassette construct, orseparately. Each of the constructs may also contain the selectablemarker gene PAT (Wohlleben et al. (1988) Gene 70:25-37) that confersresistance to the herbicide Bialaphos. Transformation is performed asfollows.

Preparation of Target Tissue: The ears are surface sterilized in 30%chlorox bleach plus 0.5% Micro detergent for 20 minutes, and rinsed twotimes with sterile water. The immature embryos are excised, placedembryo axis side down (scutellum side up), 25 embryos per plate, on 560Ymedium for 4 hours and then aligned within the 2.5-cm target zone inpreparation for bombardment.

Preparation of DNA: The DNA is precipitated onto 0.6 μm (averagediameter) gold pellets using a CaCl₂ precipitation procedure as follows:100 μl prepared gold particles in water; 10 μl (1 μg) DNA in TrisEDTAbuffer (1 μg total); 100 μl 2.5 M CaCl₂; and, 10 μl 0.1 M spermidine.

Each reagent is added sequentially to the gold particle suspension,while maintained on the multitube vortexer. The final mixture issonicated briefly and allowed to incubate under constant vortexing for10 minutes. After the precipitation period, the tubes are centrifugedbriefly, liquid removed, washed with 500 μl 100% ethanol, andcentrifuged for 30 seconds. After the liquid is removed, 105 μl 100%ethanol is added to the final gold particle pellet. For particle gunbombardment, the gold/DNA particles are briefly sonicated and 10 μlspotted onto the center of each macrocarrier and allowed to dry about 2minutes before bombardment.

The sample plates of target embryos are bombarded using approximately0.1 μg of DNA per shot using the Bio-Rad PDS-1000/He device (Bio-RadLaboratories, Hercules, Calif.) with a rupture pressure of 650 PSI, avacuum pressure of 27-28 inches of Hg, and a particle flight distance of8.5 cm. Ten aliquots are taken from each tube of prepared particles/DNA.

Following bombardment, the embryos are kept on 560Y medium for 2 days,then transferred to 560R selection medium containing 3 mg/L Bialaphos,and subcultured every 2 weeks. After approximately 10 weeks ofselection, selection-resistant callus clones are transferred to 288Jmedium to initiate plant regeneration. Following somatic embryomaturation (2-4 weeks), well-developed somatic embryos are transferredto medium for germination and transferred to the lighted culture room.Approximately 7-10 days later, developing plantlets are transferred to272V hormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto 2.5″ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to classic 600 pots (1.6 gallon) and grown to maturity.The acceptor plant will be monitored for phenotypic traits associatedwith both the site specific recombinase and the DNA construct comprisingthe target site, while the donor plant will be monitored for phenotypictraits associated with the DNA construct comprising the transfercassette.

Medium 560Y comprises 4.0 g/L N6 basal salts (SIGMA C-1416), 1.0 ml/LEriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/L thiamine HCl, 120 g/Lsucrose, 1.0 mg/L 2,4-D, and 2.88 g/L L-proline (brought to volume withD-I H₂O following adjustment to pH 5.8 with KOH); 2.0 g/L Gelrite®(added after bringing to volume with D-I H₂O); and 8.5 mg/L silvernitrate (added after sterilizing the medium and cooling to roomtemperature).

Medium 560R comprises 4.0 g/L N6 basal salts (SIGMA C-1416), 1.0 ml/LEriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/L thiamine HCl, 30.0g/L sucrose, and 2.0 mg/L 2,4-D (brought to volume with D-I H₂Ofollowing adjustment to pH 5.8 with KOH); 3.0 g/L Gelrite (added afterbringing to volume with D-I H₂O); and 0.85 mg/L silver nitrate and 3.0mg/L bialaphos (both added after sterilizing the medium and cooling toroom temperature).

Medium 288J comprises: 4.3 g/L MS salts (GIBCO 11117-074), 5.0 ml/L MSvitamins stock solution (0.100 g/L nicotinic acid, 0.02 g/L thiamineHCl, 0.10 g/L pyridoxine HCl, and 0.40 g/L glycine brought to volumewith D-I H₂O) (Murashige & Skoog (1962) Physiol Plant 15:473), 100 mg/Lmyo-inositol, 0.5 mg/L zeatin, 60 g/L sucrose, and 1.0 ml/L of 0.1 mMabscissic acid (brought to volume with D-I H₂O after adjusting to pH5.6); 3.0 g/L Gelrite (added after bringing to volume with D-I H₂O); and1.0 mg/L indoleacetic acid and 3.0 mg/L bialaphos (added aftersterilizing the medium and cooling to 60° C.).

Medium 272V comprises: 4.3 g/L MS salts (GIBCO 11117-074), 5.0 ml/L MSvitamins stock solution (0.100 g/L nicotinic acid, 0.02 g/L thiamineHCl, 0.10 g/L pyridoxine HCl, and 0.40 g/L glycine brought to volumewith D-I H₂O), 0.1 g/L myo-inositol, and 40.0 g/L sucrose (brought tovolume with D-I H₂O after adjusting pH to 5.6); and 6 g/L bacto-agar(added after bringing to volume with D-I H₂O), sterilized and cooled to60° C.

iii. Particle Bombardment of Soybean

A polynucleotide comprising a recombination site, transfer cassette,target site, other polynucleotide(s) of interest, selectable marker,and/or recombinase can be introduced into embryogenic suspensioncultures of soybean by particle bombardment using essentially themethods described in Parrott et al. (1989) Plant Cell Rep 7:615-617.This method, with modifications, is described below.

Seed is removed from pods when the cotyledons are between 3 and 5 mm inlength. The seeds are sterilized in a bleach solution (0.5%) for 15minutes after which time the seeds are rinsed with sterile distilledwater. The immature cotyledons are excised by first cutting away theportion of the seed that contains the embryo axis. The cotyledons arethen removed from the seed coat by gently pushing the distal end of theseed with the blunt end of the scalpel blade. The cotyledons are thenplaced in petri dishes (flat side up) with SB1 initiation medium (MSsalts, B5 vitamins, 20 mg/L 2,4-D, 31.5 g/L sucrose, 8 g/L TC Agar, pH5.8). The petri plates are incubated in the light (16 hr day; 75-80 μE)at 26° C. After 4 weeks of incubation the cotyledons are transferred tofresh SB1 medium. After an additional two weeks, globular stage somaticembryos that exhibit proliferative areas are excised and transferred toFN Lite liquid medium (Samoylov et al. (1998) In Vitro Cell Dev BiolPlant 34:8-13). About 10 to 12 small clusters of somatic embryos areplaced in 250 ml flasks containing 35 ml of SB172 medium. The soybeanembryogenic suspension cultures are maintained in 35 mL liquid media ona rotary shaker, 150 rpm, at 26° C. with fluorescent lights (20 μE) on a16:8 hour day/night schedule. Cultures are sub-cultured every two weeksby inoculating approximately 35 mg of tissue into 35 mL of liquidmedium.

Soybean embryogenic suspension cultures are then transformed usingparticle gun bombardment (Klein et al. (1987) Nature 327:70; U.S. Pat.No. 4,945,050). A BioRad Biolisticä PDS1000/HE instrument can be usedfor these transformations. A selectable marker gene, which is used tofacilitate soybean transformation, is a chimeric gene composed of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812), the hygromycin phosphotransferase gene from plasmid pJR225(from E. coli; Gritz et al. (1983) Gene 25:179-188) and the 3′ region ofthe nopaline synthase gene from the T-DNA of the Ti plasmid ofAgrobacterium tumefaciens.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl2(2.5 M). The particle preparation is agitated for three minutes, spun ina microfuge for 10 seconds and the supernatant removed. The DNA-coatedparticles are washed once in 400 μL 70% ethanol then resuspended in 40μL of anhydrous ethanol. The DNA/particle suspension is sonicated threetimes for one second each. Five μL of the DNA-coated gold particles arethen loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. Membrane rupture pressure is set at 1100 psi andthe chamber is evacuated to a vacuum of 28 inches mercury. The tissue isplaced approximately 8 cm away from the retaining screen, and isbombarded three times. Following bombardment, the tissue is divided inhalf and placed back into 35 ml of FN Lite medium.

Five to seven days after bombardment, the liquid medium is exchangedwith fresh medium. Eleven days post bombardment the medium is exchangedwith fresh medium containing 50 mg/mL hygromycin. This selective mediumis refreshed weekly. Seven to eight weeks post bombardment, greentransformed tissue will be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line is treated as anindependent transformation event. These suspensions are then subculturedand maintained as clusters of immature embryos, or tissue is regeneratedinto whole plants by maturation and germination of individual embryos.

C. DNA Isolation from Callus and Leaf Tissues

Putative transformation events can be screened for the presence of thetransgene. Genomic DNA is extracted from calli or leaves using amodification of the CTAB (cetyltriethylammonium bromide, Sigma H5882)method described by Stacey & Isaac (1994 In Methods in Molecular BiologyVol. 28, pp. 9-15, Ed. P. G. Isaac, Humana Press, Totowa, N.J.).Approximately 100-200 mg of frozen tissue is ground into powder inliquid nitrogen and homogenized in 1 ml of CTAB extraction buffer (2%CTAB, 0.02 M EDTA, 0.1 M TrisHCl pH 8, 1.4 M NaCl, 25 mM DTT) for 30 minat 65° C. Homogenized samples are allowed to cool at room temperaturefor 15 min before a single protein extraction with approximately 1 ml24:1 v/v chloroform:octanol is done. Samples are centrifuged for 7 minat 13,000 rpm and the upper layer of supernatant collected usingwide-mouthed pipette tips. DNA is precipitated from the supernatant byincubation in 95% ethanol on ice for 1 h. DNA threads are spooled onto aglass hook, washed in 75% ethanol containing 0.2 M sodium acetate for 10min, air-dried for 5 min and resuspended in TE buffer. Five μl RNAse Ais added to the samples and incubated at 37° C. for 1 h. Forquantification of genomic DNA, gel electrophoresis is performed using a0.8% agarose gel in 1×TBE buffer. One microlitre of each of the samplesis fractionated alongside 200, 400, 600 and 800 ng μl-1λ uncut DNAmarkers.

D. Providing the Transfer Cassette to the Acceptor Plant

A cross is performed between the donor maize plant having the transfercassette, and the acceptor maize plant having the target site.Alternatively, any transformation method can be used to provide thetransfer cassette by directly transformation into an acceptor line.Optionally, at least one of the maize plant lines is homozygous for thetarget site or transfer cassette. In addition, the acceptor plant genomemay also have stably incorporated an expression cassette encoding therecombinase. The cross is performed between the donor and the acceptorplant. Seeds from the resulting cross are collected, and targetedintegration events identified using seed priming to identify activationof the selectable marker from the transfer cassette.

E. Identification of Targeted Seed

Maize seeds were prepared as described in Example 1. Targeted seeds havethe transfer cassette integrated at the target site, activating moCAHand resistance to Dormex®. Non-targeted seed do not express moCAH andare susceptible to the Dormex® and cannot germinate. Table 4 in Example1 shows results for targeted integration events in corn inbreds N46 andP38. Samples N46-1 through P38-12 are non-transgenic controls, nogermination was observed after priming with Dormex®. Samples N46-13through N46-21 are transgenic seed, N46-21 is a transgenic controlprimed with water, N46-13 and N46-14 are from a transgenic target linecomprising PHP17797, which lacks the moCAH selectable marker, theremaining samples, P38-15 through N46-20, represent paired samples ofseed from targeted linkage and activation of moCAH selectable markerfrom the donor, to the ubiquitin promoter in the target. Seeds thatgerminated were further analyzed for cyanamide hydratase enzymeactivity, and PCR to confirm linkage of the ubi promoter to moCAH.Plants can be planted in the field or greenhouse for further analyses.

Example 4 Activating and Screening for Two Markers

The seed priming method can be extended to screen for the presence,activation and/or expression of more than one selectable marker.

A. Recombinase-Mediated Cassette Exchange Results in Activation of TwoMarker Genes in the Target Locus.

i. Target A is constructed and introduced to produce plants. Theconstruct comprises:

Target A: Rb-Ubi Pro::Ubiintron::FRT1::FLP::pinII-CaMV35S::bar:pinII-TGA::FRT5::moCAH::pinII-Lb.

After introduction of the construct, transformed cells/tissues areselected on bialaphos-containing medium and regenerated to produceTarget A plants. The expression of bar confers resistance to bialaphos.The tissue/plants can be further characterized, for example, DNA isextracted from regenerated T0 plants and subsequent T1 progeny toconfirm that the above-introduced DNA is present as a single copy usingstandard Southern analysis methods. Integration of Target A producesplants having FLP recombinase activity and bialaphos resistance (FLP⁺,BLP^(r)). The moCAH sequence (3′ to the stop codon and FRT5) is notoperably linked to the CaMV35S promoter, and therefore is not expressedin the Target A plants.

ii. Donor A is constructed and introduced to Target A plants by anymethod, including direct transformation, or by generating independentDonor A plant lines, and subsequently crossing the target and donorplants.

Donor A: Rb-CaMV35S Pro::ban:pinII-TGA-FRT1:GAT::pinII-Actin Pro::Actinintron::FRT5-Lb

If separate Donor A plants are generated, after introduction of theconstruct, transformed cells/tissues are selected onbialaphos-containing medium and regenerated to produce Donor A plants.The GAT sequence is not operably linked to a promoter, and therefore isnot expressed in the donor plants.

iii. Activation of Two Markers by Recombination Via Sexual Crossing

Target and donor plants are grown and crossed to each other. In thepresence of the recombinase, the donor cassette, containing promoterlessGAT and Actin Pro::Actin intron 5′ to FRT5, exchanges into the targetlocus, operably linking GAT to the ubiquitin promoter (Ubi Pro), andmoCah to the Actin promoter (Actin Pro). The recombined product TargetA′ locus results in expression of GAT and moCah and confers resistanceto glyphosate (GLY^(r)) and cyanamide (CYA^(r)), respectively. Progenyseed from this cross can be primed with a combination of glyphosate andcyanamide. Optionally, progeny seed could be primed sequentially in twoseparate priming matrices comprising each separate herbicide, or theprogeny seed could be primed with one herbicide, and the resistantplantlets could be leaf painted or sprayed with the second herbicide.

Progeny in which proper recombinase-mediated cassette exchange hasoccurred at both the FRT1 and FRT5 sites are FLP⁻, BLP^(r), GLY^(r) andCYA^(r). The recombination products Target A′ and Donor A′ are twoindependent loci and can be segregated away from each other in the nextgeneration(s). Further analyses, such as PCR across the recombined FRT1and FRT5 junctions, Southern analysis and/or sequencing can be used tofurther confirm that precise recombination mediated by FLP recombinaseoccurred during the cassette exchange.

B. Recombinase-Mediated Cassette Exchange Results in Activation of TwoMarker Genes in the Donor Locus.

i. Target B is constructed and introduced to produce plants. Theconstruct comprises:

Target B: Rb-FRT1-moCah::pinII/Ubi Pro::Ubiintron::FLP::pinII-CaMV35S::ban:pII-Actin Pro::Actin intron::FRT5-Lb.

After introduction of the construct, transformed cells/tissues areselected on bialaphos-containing medium and regenerated to produceTarget B plants. The expression of bar confers resistance to bialaphos.The tissue/plants can be further characterized, for example, DNA isextracted from regenerated T0 plants and subsequent T1 progeny toconfirm that the above-introduced DNA is present as a single copy usingstandard Southern analysis methods. Integration of Target B producesplants having FLP recombinase activity and bialaphos resistance (FLP⁺,BLP^(r)).

ii. Donor B is constructed and introduced to Target B plants by anymethod, including direct transformation, or by generating independentDonor B plant lines, and subsequently crossing the target and donorplants.

Donor B: Rb-CaMV35S Pro::bar::pinII-Ubi Pro::Ubiintron::FRT1::Ubi::GAT::pinII-TGA-FRT5:Actin Pro::YFP::pinII-Lb

If separate Donor B plants are generated, after introduction of theconstruct, transformed cells/tissues are selected onbialaphos-containing medium and regenerated to produce Donor B plants.

iii. Activation of Two Markers by Recombination Via Sexual Crossing

Target and donor plants are grown and crossed to each other. Donor Bcontains an active Ubi::GAT:pinII which will be inserted into the targetlocus by recombinase-mediated cassette exchange. In F1 progeny, thedesired target product cannot be identified by screening for GAT, sinceGAT is operably linked to a promoter before and after recombination.However, in the process of cassette exchange, the inactive moCah and YFPfrom the Target B locus are operably linked to promoters in therecombined Donor B′ locus, and can be used as an indication thatcassette exchange occurred. Progeny that are CYA^(r) and YFP⁺ (leavescan be measured with a hand-held OS1-FL meter; Opti-Sciences, Inc., 164Westford Rd., Tyngsboro, Mass. 01879) indicate proper cassette exchangebetween the two loci. These CYA^(r), YFP⁺ plants can be outcrossed towild-type plants, wherein the CYA^(r), YFP⁺ traits segregate as a singleunit in the progeny with Donor B′ while the GLY^(r) segregates away fromCYA^(r), YFP⁺. Putative Target B′ seed can be initially screened by seedpriming in a glyphosate solution, and then further analyzed. Forexample, the recombined Target B′ locus will further be bar⁻ (BLP^(S))and FLP⁻ due to the exchange of these cassettes into the Donor locus.Bialophos sensitivity can be assayed by leaf painting, and/or PCRanalyses across recombined FRT1 and FRT5 junctions and/or bar cassetteor other genes, as well as Southern analysis and sequencing will be usedto confirm that precise recombination mediated by FLP recombinaseoccurred during the cassette exchange.

C. Recombinase-Mediated Cassette Exchange Using Two IndependentRecombination Systems.

i. Target C is constructed and introduced to produce plants. Theconstruct comprises:

Target C: Rb-Ubi Pro::FRT1::FLP::pinII-(CaMV35Spro::bar:CaMV35Sterm)-GZ3′-pinII::Cre::loxP::Actin Pro-Lb

After introduction of the construct, transformed cells/tissues areselected on bialaphos-containing medium and regenerated to produceTarget C plants. Expression of bar confers resistance to bialaphos. Thetissue/plants can be further characterized, for example, DNA isextracted from regenerated T0 plants and subsequent T1 progeny toconfirm that the above-introduced DNA is present as a single copy usingstandard Southern analysis methods. Target C plants are FLP⁺, Cre⁺,BLP^(r).

ii. Donor C is constructed and introduced to Target C plants by anymethod, including direct transformation, or by generating independentDonor C plant lines, and subsequently crossing the target and donorplants.

Donor C: Rb-(CaMV35SPro::bar:pinII)::FRT1::GAT::pinII-GZ3′-pinII::moCah::loxP-Lb

If separate Donor C plants are generated, after introduction of theconstruct, transformed cells/tissues are selected onbialaphos-containing medium and regenerated to produce Donor C plants,which are bialaphos resistant (BLP^(r)). GAT and moCah sequences arepromoterless, and not expressed in the donor plants.

iii. Activation of Two Markers by Recombination Via Sexual Crossing

Target and donor plants are grown and crossed to each other. In thepresence of the recombinases Donor C, containing inactive GAT and moCahsequences, is exchanged into the Target C locus operably linking GAT tothe ubiquitin promoter, and moCah to the actin promoter, to generateTarget C′ conferring resistance to the herbicides glyphosate (GLY^(r))and cyanamide (CYA^(r)), respectively. Progeny seed comprising Target C′can be identified by seed priming with both herbicides, eithersimultaneously or sequentially. Optionally, seed priming is done withonly one herbicide, and the resulting seedlings can be leaf painted orsprayed with the second herbicide. The recombined loci, Target C′ andDonor C′ are independent and can be segregated away from each other inthe next generation. Further characterization, such as PCR analyses forgenes, across FRT1 and loxP junctions, as well as Southern analysis andsequencing can be used to confirm that precise recombinase-mediatedexchange occurred.

D. Recombinase-Mediated Cassette Exchange Results in Activation of OneMarker Gene in the Target Locus and One Marker Gene in the Donor Locus.

These methods and compositions can also be done in other crops, such assoybean. For example, Jack, a Glycine max (I.) Merrill cultivar can betransformed using particle bombardment.

i. Target D is constructed and introduced to produce plants. Theconstruct comprises:

Target D: SCP1 pro::FRT1::FLP::pinII-CaMV35S pro::HYG::nos term::Kti3pro-FRT6

After introduction of the construct, transformed cells/tissues areselected on hygromycin-containing medium and regenerated to produceTarget D plants. The tissue/plants can be further characterized, forexample, DNA is extracted from regenerated T0 plants and subsequent T1progeny to confirm that the above-introduced DNA is present as a singlecopy using standard Southern analysis methods. Target D plants are FLP⁺,HYG^(r).

ii. Donor D is constructed and introduced to Target D plants by anymethod, including direct transformation, or by generating independentDonor D plant lines, and subsequently crossing the target and donorplants.

Donor D: CaMV35S term-TGA-FRT1::Gm-Als::Gm-Als term-CaMV35Spro::GUS::nos term-TGA-FRT6::AmCyan1::KTI3 term

iii. Activation of Two Markers by Recombination Via Transformation

Target D plants (FLP⁺, HYG^(r)) are transformed with Donor D constructusing particle bombardment.

Recombinase-mediated exchange will produce recombined Target D′(containing SCP1 pro::FRT1::Gm-Als::Gm-Als term-CaMV35S pro::GUS::nosterm-TGA::FRT6), and recombined Donor D′ (containing CaMV35Sterm-TGA-FRT1::FLP::pinII term-CaMV35S pro::HYG::nos term::Kti3pro::FRT6::AmCyan1::Kti3 term).

After introduction of Donor D, cells/tissues can placed on mediacontaining chlorsulfuron. Alternatively, cells/tissues can be placed onmedia to generate plants and seeds. The seeds produced can be screenedby seed priming with a matrix comprising chlorsulfuron. Expression ofGm-ALS confers resistance to herbicides that inhibit acetolactatesynthase (ALS), for example sulfonylurea herbicides such aschlorsulfuron. Cells/seeds containing Target D′ will be selected for byresistance to chlorsulfuron, and can be further screened for GUS, forexample, histochemically (Jefferson, et al. (1987) EMBO J 6:3901-3907).The recombined Donor D′ is AmCyan1+ due to linkage to KTI pro, and canbe identified by screening for blue fluorescence.

E. Recombinase-Mediated Cassette Exchange Via Direct Delivery of theDonor for Activation of Two Marker Genes in the Target Locus.

i. Target E is constructed and introduced to produce plants. Theconstruct comprises:

Target E: Rb-Ubi pro::FRT1::YFP::pinII term-Ubipro::luciferase::pinII-GZ3′-term-IN2-1 term::bar:FRT5::Actin pro--Lb.After introducing Target E, cells/tissues are placed on selection mediacontaining bialophos. Transformed cells are YFP⁺, BLP^(r), Luc⁺. Cellsgrowing on selection media that are also YFP⁺ are regenerated as targetplants.

ii. Donor E is constructed and introduced to Target E plants by anymethod, including direct transformation, or by generating independentDonor E plant lines, and subsequently crossing the target and donorplants.

Target E plants can be grown and selfed, or crossed to non-transgenicplants, and immature embryos isolated. Embryos expressing YFP aretransformed using particle bombardment. Two plasmids are used, Donor E,and Plasmid R which comprises the recombinase:Donor E: Rb-TGA-FRT1::AmCyan::pinII term-Ubi pro::moCAH::pinIIterm-GZ3′-IN2-1 term::GAT::FRT5-TGA-Lb.Plasmid R: Ubi pro::FLP::pinII term.Plasmid R is typically used at a lower DNA concentration for transientexpression, rather than stable integration into the genome. If Plasmid Rintegrates, it can be removed through outcrossing and segregation of thePlasmid R locus from the Target E or E′ locus.

iii. Activation of Two Markers by Recombination Via Transformation

After bombardment with Donor E and Plasmid R, the cells/tissues canplaced on selection media containing glyphosate. Alternatively,cells/tissues can be grown on non-selective media to generate plants andseeds. At any time, Target E′ can be screened for by looking forAmCyan⁺. Progeny seed comprising Target E′ can be identified by seedpriming with both selective agents, either simultaneously orsequentially. Optionally, seed priming is done with only one selectiveagent, and the resulting seedlings can be leaf painted or sprayed withthe second selective agent. Seeds/cultures/plants expressing all threegenes can be further characterized, for example by PCR, to confirm thatrecombination occurred at the FRT1 and FRT5 junctions.

F. Recombinase-Mediated Integration Results in Activation of Two MarkerGenes in the Target Locus.

Target sites comprising two promoters operably linked to onerecombination site can be used.

i. Target F is constructed and introduced to produce plants. Theconstruct comprises:

Target F: Rb-CaMV35S pro::bar::pinII term-Ubi pro::FRT1::Actin pro(3′-5′ orientation)-Lb.After introducing Target F, cells/tissues are placed on selection mediacontaining bialophos. Transformed cells are BLP^(r) and are regeneratedas target plants.

ii. Donor F is constructed and introduced to Target F plants as acircular construct:

Donor F: -pinII term::moCAH::FRT1::GAT::term-The recombinase is provided by co-transformation with a separateconstruct comprising:Plasmid R: Ubi pro::FLP::pinII term.Plasmid R is typically used at a lower DNA concentration for transientexpression, rather than stable integration into the genome. If Plasmid Rintegrates, it can be removed through outcrossing and segregation of thePlasmid R locus from the Target F or F′ locus.

iii. Activation of Two Markers by Recombination Via Transformation

After bombardment with Donor F and Plasmid R, the cells/tissues canplaced on selection media containing glyphosate. Alternatively,cells/tissues can be grown on non-selective media to generate plants andseeds. Progeny seed comprising Target F′ can be identified by seedpriming with both herbicides, either simultaneously or sequentially.Optionally, seed priming is done with only one herbicide, and theresulting seedlings can be leaf painted or sprayed with the secondherbicide. Seeds/cultures/plants can be further characterized, forexample by PCR, to confirm the recombination junctions.

G. Recombinase-Mediated Integration Results in Activation of Two MarkerGenes in the Target Locus.

Target sites comprising two promoters operably linked to onerecombination site can be used.

i. Target G is constructed and introduced to produce plants. Theconstruct comprises:

Target G: Rb-Ubi pro::FRT1::bar::pinII term::Actin pro (3′-5′orientation)-Lb. After introducing Target F, cells/tissues are placed onselection media containing bialophos. Transformed cells are BLP^(r) andare regenerated as target plants.

ii. Donor G is constructed and introduced to Target G plants bygenerating independent Donor G plant lines using any transformationmethod, and subsequently crossing the target and donor plants. In thepresence of FLP recombinase, Donor G will be excised and circularized,and can then recombine and integrate at the Target G site. Optionally,Donor G can be constructed as a replicon, such as a viral replicon,wherein after excision and circularization, the donor will be replicatedto generate higher copy number. The recombinase can be provided ineither the target line or the donor line, and segregated away from therecombined Target G′ product.

Donor G: Rb-ubi pro::bar::pinII term-FRT1::GAT::term-pinIIterm::moCAH::FRT1-LbAfter introducing Donor G, cells/tissues are placed on selection mediacontaining bialophos. BLP^(r) events are regenerated into Donor G plantsand used for crossing.

iii. Activation of Two Markers by Recombination Via Crossing

In the presence of FLP recombinase, Donor G will be excised andcircularized, and can then recombine and integrate at the Target G site.Optionally, Donor G can be constructed as a replicon, such as a viralreplicon, wherein after excision and circularization, the donor will bereplicated to generate higher copy number. The recombinase can beprovided in either the target line or the donor line, and segregatedaway from the recombined Target G′ product. Optionally, thepolynucleotide encoding FLP is linked to an inducible or tissue-specificpromoter, in order to control FLP availability, and possibly minimizethe reverse reaction. Progeny seed comprising Target G′ can beidentified by seed priming with both herbicides, either simultaneouslyor sequentially. Optionally, seed priming is done with only oneherbicide, and the resulting seedlings can be leaf painted or sprayedwith the second herbicide. Seeds/cultures/plants can be furthercharacterized, for example by PCR, to confirm the recombinationjunctions.

The articles “a” and “an” refer to one or more than one of thegrammatical object of the article. By way of example, “an element” meansone or more element.

All book, journal, patent publications and grants mentioned in thespecification are indicative of the level of those skilled in the art.All publications and patent applications are herein incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, certain changes and modifications may be practiced withinthe scope of the appended claims.

1. A method to identify a plant having a DNA construct stablyincorporated in its genome comprising: (a) providing a population ofseeds, wherein at least one seed in the population has stablyincorporated into its genome the DNA construct comprising the followingoperably linked components in the following order: a promoter active inthe seed, a first recombination site, a polynucleotide encoding a firstselection marker that confers resistance to a selective agent, and asecond recombination site; (b) contacting the population of seeds with apriming matrix comprising an effective concentration of the selectionagent for a time sufficient to produce a population of primed seeds;and, (c) incubating the population of primed seed under germinationconditions to produce germinated seeds comprising the DNA construct,thereby identifying plants having the DNA construct stably incorporatedinto their genome.
 2. The method of claim 1, wherein the first and thesecond recombination sites are dissimilar and non-recombinogenic witheach other.
 3. The method of claim 1, wherein the selective agentcomprises an herbicide, a growth regulator, or an antibiotic.
 4. Themethod of claim 3, wherein the selective agent is selected from thegroup consisting of glufosinate, phosphinothricin, glyphosate,bromoxynil, methotrexate, imidazolinones, sulfonylureas, cyanamide,kanamycin, G418, neomycin, and hygromycin B.
 5. The method of claim 3,wherein the first selection marker is selected from the group consistingof Bar, phosphinothricin acetyltransferase (PAT), glyphosateoxidoreductase (GOX), 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS), CP4, glyphosate N-acetyltransferase (GAT), bromoxynil nitrilase(BXN), dihydrofolate reductase, acetolactate synthase (ALS), cyanamidehydratase (CAH), neomycin phosphotransferase (nptII), aminoglycoside3′-phosphotransferase (APH3′ II), and hygromycin phosphotransferase(hph).
 6. The method of claim 5, wherein the polynucleotide encoding thefirst selection marker has been synthesized using maize preferredcodons.
 7. The method of claim 1, wherein the DNA construct comprises inthe following order: a first expression unit comprising the promoteractive in the seed operably linked to the first recombination siteoperably linked to the polynucleotide encoding the first selectionmarker; and a second expression unit comprising in the following order,a second promoter active in the plant operably linked to apolynucleotide of interest, and the second recombination site.
 8. Themethod of claim 7, wherein the polynucleotide of interest encodes asecond selection marker, wherein the second selection marker is distinctfrom the first selection marker by having a different selection means.9. The method of claim 1, wherein the plant further has stablyincorporated into its genome a polynucleotide encoding a site specificrecombinase.
 10. The method of claim 9, wherein the site specificrecombinase comprises a FLP recombinase, a Cre recombinase, a lambdaintegrase, a SSV1 integrase, or a phiC31 integrase.
 11. The method ofclaim 1, wherein at least one of the first or the second recombinationsites are selected from the group consisting of a lox site, a mutant loxsite, a FRT site, a mutant FRT site, an att site, and a mutant att site.12. The method of claim 1, wherein the plant is maize, wheat, rice,sorghum, rye, oat, barley, millet, Brassica, alfalfa, sunflower,safflower, soybean, tobacco, cotton, or Arabidopsis.
 13. A method ofpriming a seed comprising (a) providing a seed having stablyincorporated into its genome a DNA construct comprising, in thefollowing order, a promoter active in the seed operably linked to afirst recombination site operably linked to a polynucleotide encoding aselection marker that confers resistance to a selective agent; and (b)contacting the seed with a priming matrix comprising an effectiveconcentration of the selective agent for a time sufficient to produce aprimed seed.
 14. The method of claim 13, wherein the DNA constructcomprises in the following order the promoter operably linked to thefirst recombination site operably linked the polynucleotide encoding theselection marker, and a second recombination site.
 15. The method ofclaim 14, wherein the first and the second recombination sites aredissimilar and non-recombinogenic with each other.
 16. A compositioncomprising a seed having stably incorporated into its genome a DNAconstruct comprising, in the following order, a promoter active in theseed operably linked to a first recombination site operably linked to apolynucleotide encoding a selection marker that confers resistance to aselective agent; and, a priming matrix comprising an effectiveconcentration of the selective agent.
 17. The composition of claim 16,wherein the DNA construct comprises in the following order, the promoteroperably linked to the first recombination site operably linked to thepolynucleotide encoding the selection marker, and a second recombinationsite.
 18. The composition of claim 17, wherein the first and the secondrecombination sites are dissimilar and non-recombinogenic with eachother.
 19. The composition claim 16, wherein the DNA construct furthercomprises a polynucleotide of interest.
 20. The composition of any oneof claim 16, wherein the DNA construct further comprises a secondselection marker.
 21. The composition of claim 20, wherein the secondselection marker confers resistance to a second selective agent.
 22. Thecomposition of claim 20, wherein the priming matrix further comprisesthe second selective agent.
 23. The composition of claim 16, wherein theseed is from a plant selected from the group consisting of maize, wheat,rice, sorghum, rye, oat, barley, millet, Brassica, alfalfa, sunflower,safflower, soybean, tobacco, cotton, and Arabidopsis.